Dual variable domain immunoglobulins and uses thereof

ABSTRACT

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application claiming priority toU.S. Provisional Application Ser. No. 61/130,776, filed Jun. 3, 2008 andU.S. Provisional Application Ser. No. 61/132,951, filed Jun. 24, 2008,the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to multivalent and multispecific bindingproteins, methods of making, and specifically to their uses in the,diagnosis, prevention and/or treatment of acute and chronic inflammatorydiseases, cancer, and other diseases.

BACKGROUND OF THE INVENTION

Engineered proteins, such as multispecific antibodies capable of bindingtwo or more antigens are known in the art. Such multispecific bindingproteins can be generated using cell fusion, chemical conjugation, orrecombinant DNA techniques.

Bispecific antibodies have been produced using quadroma technology (seeMilstein, C. and A. C. Cuello (1983) Nature 305(5934):537-40) based onthe somatic fusion of two different hybridoma cell lines expressingmurine monoclonal antibodies (mAbs) with the desired specificities ofthe bispecific antibody. Because of the random pairing of two differentimmunoglobulin (Ig) heavy and light chains within the resultinghybrid-hybridoma (or quadroma) cell line, up to ten different Ig speciesare generated, of which only one is the functional bispecific antibody.The presence of mis-paired by-products, and significantly reducedproduction yields, means sophisticated purification procedures arerequired.

Bispecific antibodies can also be produced by chemical conjugation oftwo different mAbs (see Staerz, U. D., et al. (1985) Nature 314(6012):628-31). This approach does not yield homogeneous preparation. Otherapproaches have used chemical conjugation of two different mAbs orsmaller antibody fragments (see Brennan, M., et al. (1985) Science229(4708): 81-3).

Another method used to produce bispecific antibodies is the coupling oftwo parental antibodies with a hetero-bifunctional crosslinker, but theresulting bispecific antibodies suffer from significant molecularheterogeneity because reaction of the crosslinker with the parentalantibodies is not site-directed. To obtain more homogeneous preparationsof bispecific antibodies two different Fab fragments have beenchemically crosslinked at their hinge cysteine residues in asite-directed manner (see Glennie, M. J., et al. (1987) J. Immunol.139(7): 2367-75). But this method results in Fab′2 fragments, not fullIgG molecule.

A wide variety of other recombinant bispecific antibody formats havebeen developed (see Kriangkum, J., et al. (2001) Biomol. Eng. 18(2):31-40). Amongst them tandem single-chain Fv molecules and diabodies, andvarious derivatives thereof, are the most widely used. Routinely,construction of these molecules starts from two single-chain Fv (scFv)fragments that recognize different antigens (see Economides, A. N., etal. (2003) Nat. Med. 9(1): 47-52). Tandem scFv molecules (taFv)represent a straightforward format simply connecting the two scFvmolecules with an additional peptide linker. The two scFv fragmentspresent in these tandem scFv molecules form separate folding entities.Various linkers can be used to connect the two scFv fragments andlinkers with a length of up to 63 residues (see Nakanishi, K., et al.(2001) Ann. Rev. Immunol. 19: 423-74). Although the parental scFvfragments can normally be expressed in soluble form in bacteria, it is,however, often observed that tandem scFv molecules form insolubleaggregates in bacteria. Hence, refolding protocols or the use ofmammalian expression systems are routinely applied to produce solubletandem scFv molecules. In a recent study, in vivo expression bytransgenic rabbits and cattle of a tandem scFv directed against CD28 anda melanoma-associated proteoglycan was reported (see Gracie, J. A., etal. (1999) J. Clin. Invest. 104(10): 1393-401). In this construct, thetwo scFv molecules were connected by a CH1 linker and serumconcentrations of up to 100 mg/L of the bispecific antibody were found.Various strategies including variations of the domain order or usingmiddle linkers with varying length or flexibility were employed to allowsoluble expression in bacteria. A few studies have now reportedexpression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al. (2000) J. Immunol. 164(12): 6495-502; Ito, A., et al. (2003)J. Immunol. 170(9): 4802-9; Karni, A., et al. (2002) J. Neuroimmunol.125(1-2): 134-40) using either a very short Ala3 linker or longglycine/serine-rich linkers. In another recent study, phage display of atandem scFv repertoire containing randomized middle linkers with alength of 3 or 6 residues was employed to enrich for those moleculesthat are produced in soluble and active form in bacteria. This approachresulted in the isolation of a tandem scFv molecule with a 6 amino acidresidue linker (see Arndt, M. and J. Krauss (2003) Methods Mol. Biol.207: 305-21). It is unclear whether this linker sequence represents ageneral solution to the soluble expression of tandem scFv molecules.Nevertheless, this study demonstrated that phage display of tandem scFvmolecules in combination with directed mutagenesis is a powerful tool toenrich for these molecules, which can be expressed in bacteria in anactive form.

Bispecific diabodies (Db) utilize the diabody format for expression.Diabodies are produced from scFv fragments by reducing the length of thelinker connecting the VH and VL domain to approximately 5 residues (seePeipp, M. and T. Valerius (2002) Biochem. Soc. Trans. 30(4): 507-11).This reduction of linker size facilitates dimerization of twopolypeptide chains by crossover pairing of the VH and VL domains.Bispecific diabodies are produced by expressing, two polypeptide chainswith, either the structure VHA-VLB and VHB-VLA (VH-VL configuration), orVLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A largevariety of different bispecific diabodies have been produced in the pastand most of them are expressed in soluble form in bacteria. However, arecent comparative study demonstrates that the orientation of thevariable domains can influence expression and formation of activebinding sites (see Mack, M. et al. (1995) Proc. Natl. Acad. Sci. USA92(15): 7021-5). Nevertheless, soluble expression in bacteria representsan important advantage over tandem scFv molecules. However, since twodifferent polypeptide chains are expressed within a single cell inactivehomodimers can be produced together with active heterodimers. Thisnecessitates the implementation of additional purification steps inorder to obtain homogenous preparations of bispecific diabodies. Oneapproach to force the generation of bispecific diabodies is theproduction of knob-into-hole diabodies (see Holliger, P., T. Prospero,and G. Winter (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-8.18). Thisapproach was demonstrated for a bispecific diabody directed against HER2and CD3. A large knob was introduced in the VH domain by exchangingVa137 with Phe and Leu45 with Trp and a complementary hole was producedin the VL domain by mutating Phe98 to Met and Tyr87 to Ala, either inthe anti-HER2 or the anti-CD3 variable domains. By using this approachthe production of bispecific diabodies could be increased from 72% bythe parental diabody to over 90% by the knob-into-hole diabody.Importantly, production yields only slightly decrease as a result ofthese mutations. However, a reduction in antigen-binding activity wasobserved for several constructs. Thus, this rather elaborate approachrequires the analysis of various constructs in order to identify thosemutations that produce heterodimeric molecule with unaltered bindingactivity. In addition, such approach requires mutational modification ofthe immunoglobulin sequence at the constant region, thus creatingnon-native and non-natural form of the antibody sequence, which mayresult in increased immunogenicity, poor in vivo stability, as well asundesirable pharmacokinetics.

Single-chain diabodies (scDb) represent an alternative strategy forimproving the formation of bispecific diabody-like molecules (seeHolliger, P. and G. Winter (1997) Cancer Immunol. Immunother. 45(3-4):128-30; Wu, A. M., et al. (1996) Immunotechnology 2(1): p. 21-36).Bispecific single-chain diabodies are produced by connecting the twodiabody-forming polypeptide chains with an additional middle linker witha length of approximately 15 amino acid residues. Consequently, allmolecules with a molecular weight corresponding to monomericsingle-chain diabodies (50-60 kDa) are bispecific. Several studies havedemonstrated that bispecific single chain diabodies are expressed inbacteria in soluble and active form with the majority of purifiedmolecules present as monomers (see Holliger, P. and G. Winter (1997)Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A. M., et al. (1996)Immunotechnol. 2(1): 21-36; Pluckthun, A. and P. Pack (1997)Immunotechnol. 3(2): 83-105; Ridgway, J. B., et al. (1996) ProteinEngin. 9(7): 617-21). Thus, single-chain diabodies combine theadvantages of tandem scFvs (all monomers are bispecific) and diabodies(soluble expression in bacteria).

More recently diabodies have been fused to Fc to generate more Ig-likemolecules, named di-diabodies (see Lu, D., et al. (2004) J. Biol. Chem.279(4): 2856-65). In addition, multivalent antibody construct comprisingtwo Fab repeats in the heavy chain of an IgG and capable of binding fourantigen molecules has been described (see WO 0177342A1, and Miller, K.,et al. (2003) J. Immunol. 170(9): 4854-61).

There is a need in the art for improved multivalent binding proteinscapable of binding two or more antigens. U.S. patent application Ser.No. 11/507,050 provides a novel family of binding proteins capable ofbinding two or more antigens with high affinity, which are called dualvariable domain immunoglobulins (DVD-Ig™). The present inventionprovides further novel binding proteins capable of binding two or moreantigens.

SUMMARY OF THE INVENTION

This invention pertains to multivalent binding proteins capable ofbinding two or more antigens. The present invention provides a novelfamily of binding proteins capable of binding two or more antigens withhigh affinity.

In one embodiment the invention provides a binding protein comprising apolypeptide chain, wherein the polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is asecond variable domain, C is a constant domain, X1 represents an aminoacid or polypeptide, X2 represents an Fc region and n is 0 or 1. In anembodiment the VD1 and VD2 in the binding protein are heavy chainvariable domains. In another embodiment, the heavy chain variable domainis selected from the group consisting of a murine heavy chain variabledomain, a human heavy chain variable domain, a CDR grafted heavy chainvariable domain, and a humanized heavy chain variable domain. In yetanother, embodiment VD1 and VD2 are capable of binding the same antigen.In another embodiment VD1 and VD2 are capable of binding differentantigens. In still another embodiment, C is a heavy chain constantdomain. For example, X1 is a linker with the proviso that X1 is not CH1.For example, X1 is a linker selected from the group consisting ofAKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO:5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO:10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ IDNO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15);QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP(SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20);ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS(SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQID NO: 25); and GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an embodiment, X2 isan Fc region. In another embodiment, X2 is a variant Fc region.

In an embodiment the binding protein disclosed herein comprises apolypeptide chain, wherein the polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variabledomain, VD2 is a second heavy chain variable domain, C is a heavy chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 is an Fc region.

In an embodiment, VD1 and VD2 in the binding protein are light chainvariable domains. In an embodiment, the light chain variable domain isselected from the group consisting of a murine light chain variabledomain, a human light chain variable domain, a CDR grafted light chainvariable domain, and a humanized light chain variable domain. In oneembodiment VD1 and VD2 are capable of binding the same antigen. Inanother embodiment VD1 and VD2 are capable of binding differentantigens. In an embodiment, C is a light chain constant domain. Inanother embodiment, X1 is a linker with the proviso that X1 is not CL1.In an embodiment, X1 is a linker selected from the group consisting ofAKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO:5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO:10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ IDNO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15);QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP(SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20);ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS(SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQID NO: 25); and GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an embodiment, thebinding protein does not comprise X2.

In an embodiment, both the variable heavy and variable light chaincomprise the same linker. In another embodiment, the variable heavy andvariable light chain comprise different linkers. In another embodiment,both the variable heavy and variable light chain comprise a short (about6 amino acids) linker. In another embodiment, both the variable heavyand variable light chain comprise a long (greater than 6 amino acids)linker. In another embodiment, the variable heavy chain comprises ashort linker and the variable light chain comprises a long linker. Inanother embodiment, the variable heavy chain comprises a long linker andthe variable light chain comprises a short linker.

In an embodiment the binding protein disclosed herein comprises apolypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 does not comprise an Fc region.

In another embodiment the invention provides a binding proteincomprising two polypeptide chains, wherein said first polypeptide chaincomprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chainvariable domain, VD2 is a second heavy chain variable domain, C is aheavy chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 is an Fc region; and said second polypeptide chaincomprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chainvariable domain, VD2 is a second light chain variable domain, C is alight chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 does not comprise an Fc region. In a particularembodiment, the Dual Variable Domain (DVD) binding protein comprisesfour polypeptide chains wherein the first two polypeptide chainscomprises VD1-(X1)n-VD2-C-(X2)n, respectively wherein VD1 is a firstheavy chain variable domain, VD2 is a second heavy chain variabledomain, C is a heavy chain constant domain, X1 is a linker with theproviso that it is not CH1, and X2 is an Fc region; and the second twopolypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n respectively, whereinVD1 is a first light chain variable domain, VD2 is a second light chainvariable domain, C is a light chain constant domain, X1 is a linker withthe proviso that it is not CH1, and X2 does not comprise an Fc region.Such a Dual Variable Domain (DVD) protein has four antigen bindingsites.

In another embodiment the binding proteins disclosed herein are capableof binding one or more targets. In an embodiment, the target is selectedfrom the group consisting of cytokines, cell surface proteins, enzymesand receptors. In another embodiment, the binding protein is capable ofmodulating a biological function of one or more targets. In anotherembodiment, the binding protein is capable of neutralizing one or moretargets. The binding protein of the invention is capable of bindingcytokines selected from the group consisting of lymphokines, monokines,polypeptide hormones, receptors, or tumor markers. For example, theDVD-Ig of the invention is capable of binding two or more of thefollowing: Amyloid beta (Abeta) (seq. 1), Abeta (seq. 2), Abeta (seq.3), Tumor Necrosis Factor alpha (TNF-α), interleukin 1β (IL-1β),Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2(IGF2), Interleukin 18 (IL-18), Interleukin 6 (IL-6), Receptor forAdvanced Glycation Endproducts (RAGE), Nerve Growth Factor (NGF),Epidermal Growth Factor Receptor (EGFR) (seq. 1), EGFR (seq. 2), CD-20,and Repulsive Guidance Molecule (RGMA) (see also Table 2). In a specificembodiment the binding protein is capable of binding pairs of targetsselected from the group consisting of Abeta (seq. 1) and Abeta (seq. 3);TNF-α and Abeta (seq. 2); IL-1β and Abeta (seq. 2); Abeta (seq. 1) andAbeta (seq. 2); IGF1,2 and Abeta (seq. 2); Abeta (seq. 2) and IL-18;IL-6 and Abeta (seq. 2); TNF-α and Abeta (seq.3); IL-1β and Abeta (seq.3); Abeta (seq. 1) and Abeta (seq. 3); IGF1,2 and Abeta (seq. 3); Abeta(seq. 3) and IL-18; IL-6 and Abeta (seq. 3); TNF-α and Abeta (seq. 1);IL-1β and Abeta (seq. 1); IGF1,2 and Abeta (seq. 1); Abeta (seq. 1) andIL-18; IL-6 and Abeta (seq. 1); Abeta (seq. 1) and RAGE; NGF and IL-18;NGF and IL-1β; NGF and IL-6; TNF-α and EGFR (seq. 2); TNF-α and RAGE;CD-20 and EGFR (seq. 1); CD-20 and EGFR (seq. 2); RGMA and TNF-α (seeExamples 2.1 to 2.27).

In an embodiment, the binding protein capable of binding Abeta (seq. 1)and Abeta (seq. 3) comprises a DVD heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 62 and SEQ ID NO. 64;and a DVD light chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 63 and SEQ ID NO. 65. In an embodiment, thebinding protein capable of binding Abeta (seq. 1) and Abeta (seq. 3)comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 62 and aDVD light chain amino acid sequence of SEQ ID NO: 63. In anotherembodiment, the binding protein capable of binding Abeta (seq. 1) andAbeta (seq. 3) has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 64 and a DVD light chain amino acidsequence of SEQ ID NO: 65.

In an embodiment, the binding protein capable of binding TNF-α and Abeta(seq. 2) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 66 and SEQ ID NO. 68; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 67 and SEQ ID NO. 69. In an embodiment, the binding protein capableof binding TNF-α and Abeta (seq. 2) comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 66 and a DVD light chain amino acid sequenceof SEQ ID NO: 67. In another embodiment, the binding protein capable ofbinding TNF-α and Abeta (seq. 2) has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 68 and a DVD lightchain amino acid sequence of SEQ ID NO: 69.

In an embodiment, the binding protein capable of binding IL-1β and Abeta(seq. 2) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 70 and SEQ ID NO. 72; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 71 and SEQ ID NO. 73. In an embodiment, the binding protein capableof binding IL-1β and Abeta (seq. 2) comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 70 and a DVD light chain amino acid sequenceof SEQ ID NO: 71. In another embodiment, the binding protein capable ofbinding IL-1β and Abeta (seq. 2) has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 72 and a DVD lightchain amino acid sequence of SEQ ID NO: 73.

In an embodiment, the binding protein capable of binding Abeta (seq. 1)and Abeta (seq. 2) comprises a DVD heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 74 and SEQ ID NO. 76;and a DVD light chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 75 and SEQ ID NO. 77. In an embodiment, thebinding protein capable of binding Abeta (seq. 1) and Abeta (seq. 2)comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 74 and aDVD light chain amino acid sequence of SEQ ID NO: 75. In anotherembodiment, the binding protein capable of binding Abeta (seq. 1) andAbeta (seq. 2) has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 76 and a DVD light chain amino acidsequence of SEQ ID NO: 77.

In an embodiment, the binding protein capable of binding IGF1,2 andAbeta (seq. 2) comprises a DVD heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO. 78 and SEQ ID NO. 80; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 79 and SEQ ID NO. 81. In an embodiment, the binding proteincapable of binding IGF1,2 and Abeta (seq. 2) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 78 and a DVD light chain amino acidsequence of SEQ ID NO: 79. In another embodiment, the binding proteincapable of binding IGF1,2 and Abeta (seq. 2) has a reverse orientationand comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 80 anda DVD light chain amino acid sequence of SEQ ID NO: 81.

In an embodiment, the binding protein capable of binding Abeta (seq. 2)and IL-18 comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 82 and SEQ ID NO. 84; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 83 and SEQ ID NO. 85. In an embodiment, the binding protein capableof binding Abeta (seq. 2) and IL-18 comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 82 and a DVD light chain amino acid sequenceof SEQ ID NO: 83. In another embodiment, the binding protein capable ofbinding Abeta (seq. 2) and IL-18 has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 84 and a DVD lightchain amino acid sequence of SEQ ID NO: 85.

In an embodiment, the binding protein capable of binding IL-6 and Abeta(seq. 2) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 86 and SEQ ID NO. 88; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 87 and SEQ ID NO. 89. In an embodiment, the binding protein capableof binding IL-6 and Abeta (seq. 2) comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 86 and a DVD light chain amino acid sequenceof SEQ ID NO: 87. In another embodiment, the binding protein capable ofbinding IL-6 and Abeta (seq. 2) has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 88 and a DVD lightchain amino acid sequence of SEQ ID NO: 89.

In an embodiment, the binding protein capable of binding TNF-α and Abeta(seq.3) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 90 and SEQ ID NO. 92; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 91 and SEQ ID NO. 93. In an embodiment, the binding protein capableof binding TNF-α and Abeta (seq.3) comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 90 and a DVD light chain amino acid sequenceof SEQ ID NO: 91. In another embodiment, the binding protein capable ofbinding TNF-α and Abeta (seq.3) has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 92 and a DVD lightchain amino acid sequence of SEQ ID NO: 93.

In an embodiment, the binding protein capable of binding IL-1β and Abeta(seq. 3) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 94 and SEQ ID NO. 96; and a DVD lightchain amino acid sequence selected from the group consisting of SEQ IDNO. 95 and SEQ ID NO. 97. In an embodiment, the binding protein capableof binding IL-1β and Abeta (seq. 3) comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 94 and a DVD light chain amino acid sequenceof SEQ ID NO: 95. In another embodiment, the binding protein capable ofbinding IL-1β and Abeta (seq. 3) has a reverse orientation and comprisesa DVD heavy chain amino acid sequence of SEQ ID NO. 96 and a DVD lightchain amino acid sequence of SEQ ID NO: 97.

In an embodiment, the binding protein capable of binding Abeta (seq. 1)and Abeta (seq. 3) comprises a DVD heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 98 and SEQ ID NO. 100;and a DVD light chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 99 and SEQ ID NO. 101. In an embodiment, thebinding protein capable of binding Abeta (seq. 1) and Abeta (seq. 3)comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 98 and aDVD light chain amino acid sequence of SEQ ID NO: 99. In anotherembodiment, the binding protein capable of binding Abeta (seq. 1) andAbeta (seq. 3) has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 100 and a DVD light chain amino acidsequence of SEQ ID NO: 101.

In an embodiment, the binding protein capable of binding IGF1,2 andAbeta (seq. 3) comprises a DVD heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO. 102 and SEQ ID NO. 104; and aDVD light chain amino acid sequence selected from the group consistingof SEQ ID NO. 103 and SEQ ID NO. 105. In an embodiment, the bindingprotein capable of binding IGF1,2 and Abeta (seq. 3) comprises a DVDheavy chain amino acid sequence of SEQ ID NO. 102 and a DVD light chainamino acid sequence of SEQ ID NO: 103. In another embodiment, thebinding protein capable of binding IGF1,2 and Abeta (seq. 3) has areverse orientation and comprises a DVD heavy chain amino acid sequenceof SEQ ID NO. 104 and a DVD light chain amino acid sequence of SEQ IDNO: 105.

In an embodiment, the binding protein capable of binding Abeta (seq. 3)and IL-18 comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 106 and SEQ ID NO. 108; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 107 and SEQ ID NO. 109. In an embodiment, the binding proteincapable of binding Abeta (seq. 3) and IL-18 comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 106 and a DVD light chain amino acidsequence of SEQ ID NO: 107.

In another embodiment, the binding protein capable of binding Abeta(seq. 3) and IL-18 has a reverse orientation and comprises a DVD heavychain amino acid sequence of SEQ ID NO. 108 and a DVD light chain aminoacid sequence of SEQ ID NO: 109.

In an embodiment, the binding protein capable of binding IL-6 and Abeta(seq. 3) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 110 and SEQ ID NO. 112; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 111 and SEQ ID NO. 113. In an embodiment, the binding proteincapable of binding IL-6 and Abeta (seq. 3) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 110 and a DVD light chain amino acidsequence of SEQ ID NO: 111. In another embodiment, the binding proteincapable of binding IL-6 and Abeta (seq. 3) has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 112 and aDVD light chain amino acid sequence of SEQ ID NO: 113.

In an embodiment, the binding protein capable of binding TNF-α and Abeta(seq. 1) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 114 and SEQ ID NO. 116; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 115 and SEQ ID NO. 117. In an embodiment, the binding proteincapable of binding TNF-α and Abeta (seq. 1) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 114 and a DVD light chain amino acidsequence of SEQ ID NO: 115. In another embodiment, the binding proteincapable of binding TNF-α and Abeta (seq. 1) has a reverse orientationand comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 116and a DVD light chain amino acid sequence of SEQ ID NO: 117.

In an embodiment, the binding protein capable of binding IL-1β and Abeta(seq. 1) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 118 and SEQ ID NO. 120; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 119 and SEQ ID NO. 121. In an embodiment, the binding proteincapable of binding IL-1β and Abeta (seq. 1) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 118 and a DVD light chain amino acidsequence of SEQ ID NO: 119. In another embodiment, the binding proteincapable of binding IL-1β and Abeta (seq. 1) has a reverse orientationand comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 120and a DVD light chain amino acid sequence of SEQ ID NO: 121.

In an embodiment, the binding protein capable of binding IGF1,2 andAbeta (seq. 1) comprises a DVD heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO. 122 and SEQ ID NO. 124; and aDVD light chain amino acid sequence selected from the group consistingof SEQ ID NO. 123 and SEQ ID NO. 125. In an embodiment, the bindingprotein capable of binding IGF1,2 and Abeta (seq. 1) comprises a DVDheavy chain amino acid sequence of SEQ ID NO. 122 and a DVD light chainamino acid sequence of SEQ ID NO: 123. In another embodiment, thebinding protein capable of binding IGF1,2 and Abeta (seq. 1) has areverse orientation and comprises a DVD heavy chain amino acid sequenceof SEQ ID NO. 124 and a DVD light chain amino acid sequence of SEQ IDNO: 125.

In an embodiment, the binding protein capable of binding Abeta (seq. 1)and IL-18 comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 126 and SEQ ID NO. 128; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 127 and SEQ ID NO. 129. In an embodiment, the binding proteincapable of binding Abeta (seq. 1) and IL-18 comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 126 and a DVD light chain amino acidsequence of SEQ ID NO: 127. In another embodiment, the binding proteincapable of binding Abeta (seq. 1) and IL-18 has a reverse orientationand comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 128and a DVD light chain amino acid sequence of SEQ ID NO: 129.

In an embodiment, the binding protein capable of binding IL-6 and Abeta(seq. 1) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 130 and SEQ ID NO. 132; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 131 and SEQ ID NO. 133. In an embodiment, the binding proteincapable of binding IL-6 and Abeta (seq. 1) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 130 and a DVD light chain amino acidsequence of SEQ ID NO: 131. In another embodiment, the binding proteincapable of binding IL-6 and Abeta (seq. 1) has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 132 and aDVD light chain amino acid sequence of SEQ ID NO: 133.

In an embodiment, the binding protein capable of binding Abeta (seq. 1)and RAGE comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 134 and SEQ ID NO. 136; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 135 and SEQ ID NO. 137. In an embodiment, the binding proteincapable of binding Abeta (seq. 1) and RAGE comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 134 and a DVD light chain amino acidsequence of SEQ ID NO: 135. In another embodiment, the binding proteincapable of binding Abeta (seq. 1) and RAGE has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 136 and aDVD light chain amino acid sequence of SEQ ID NO: 137.

In an embodiment, the binding protein capable of binding NGF and IL-18comprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 138 and SEQ ID NO. 140; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 139and SEQ ID NO. 141. In an embodiment, the binding protein capable ofbinding NGF and IL-18 comprises a DVD heavy chain amino acid sequence ofSEQ ID NO. 138 and a DVD light chain amino acid sequence of SEQ ID NO:139. In another embodiment, the binding protein capable of binding NGFand IL-18 has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 140 and a DVD light chain amino acidsequence of SEQ ID NO: 141.

In an embodiment, the binding protein capable of binding NGF and IL-1βcomprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 142 and SEQ ID NO. 144; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 143and SEQ ID NO. 145. In an embodiment, the binding protein capable ofbinding NGF and IL-1β comprises a DVD heavy chain amino acid sequence ofSEQ ID NO. 142 and a DVD light chain amino acid sequence of SEQ ID NO:143. In another embodiment, the binding protein capable of binding NGFand IL-1β has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 144 and a DVD light chain amino acidsequence of SEQ ID NO: 145.

In an embodiment, the binding protein capable of binding NGF and IL-6comprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 146 and SEQ ID NO. 148; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 147and SEQ ID NO. 149. In an embodiment, the binding protein capable ofbinding NGF and IL-6 comprises a DVD heavy chain amino acid sequence ofSEQ ID NO. 146 and a DVD light chain amino acid sequence of SEQ ID NO:147. In another embodiment, the binding protein capable of binding NGFand IL-6 has a reverse orientation and comprises a DVD heavy chain aminoacid sequence of SEQ ID NO. 148 and a DVD light chain amino acidsequence of SEQ ID NO: 149.

In an embodiment, the binding protein capable of binding TNF-α and EGFR(seq. 2) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 150 and SEQ ID NO. 152; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 151 and SEQ ID NO. 153. In an embodiment, the binding proteincapable of binding TNF-α and EGFR (seq. 2) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 150 and a DVD light chain amino acidsequence of SEQ ID NO: 151. In another embodiment, the binding proteincapable of binding TNF-α and EGFR (seq. 2) has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 152 and aDVD light chain amino acid sequence of SEQ ID NO: 153.

In an embodiment, the binding protein capable of binding TNF-α and RAGEcomprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 154 and SEQ ID NO. 156; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 155and SEQ ID NO. 157. In an embodiment, the binding protein capable ofbinding TNF-α and RAGE comprises a DVD heavy chain amino acid sequenceof SEQ ID NO. 154 and a DVD light chain amino acid sequence of SEQ IDNO: 155. In another embodiment, the binding protein capable of bindingTNF-α and RAGE has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 156 and a DVD light chain amino acidsequence of SEQ ID NO: 157.

In an embodiment, the binding protein capable of binding CD-20 and EGFR(seq. 1) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 158 and SEQ ID NO. 160; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 159 and SEQ ID NO. 161. In an embodiment, the binding proteincapable of binding CD-20 and EGFR (seq. 1) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 158 and a DVD light chain amino acidsequence of SEQ ID NO: 159. In another embodiment, the binding proteincapable of binding CD-20 and EGFR (seq. 1) has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 160 and aDVD light chain amino acid sequence of SEQ ID NO: 161.

In an embodiment, the binding protein capable of binding CD-20 and EGFR(seq. 2) comprises a DVD heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO. 162 and SEQ ID NO. 164; and a DVDlight chain amino acid sequence selected from the group consisting ofSEQ ID NO. 163 and SEQ ID NO. 164. In an embodiment, the binding proteincapable of binding CD-20 and EGFR (seq. 2) comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 162 and a DVD light chain amino acidsequence of SEQ ID NO: 163. In another embodiment, the binding proteincapable of binding CD-20 and EGFR (seq. 2) has a reverse orientation andcomprises a DVD heavy chain amino acid sequence of SEQ ID NO. 164 and aDVD light chain amino acid sequence of SEQ ID NO: 165.

In an embodiment, the binding protein capable of binding RGMA and TNF-αcomprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 166 and SEQ ID NO. 168; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 167and SEQ ID NO. 169. In an embodiment, the binding protein capable ofbinding RGMA and TNF-α comprises a DVD heavy chain amino acid sequenceof SEQ ID NO. 166 and a DVD light chain amino acid sequence of SEQ IDNO: 167. In another embodiment, the binding protein capable of bindingRGMA and TNF-α has a reverse orientation and comprises a DVD heavy chainamino acid sequence of SEQ ID NO. 168 and a DVD light chain amino acidsequence of SEQ ID NO: 169.

In another embodiment the invention provides a binding proteincomprising a polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second heavy chain variable domain obtained from asecond parent antibody or antigen binding portion thereof; C is a heavychain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n is an Fcregion, wherein said (X2)n is either present or absent. In anembodiment, the Fc region is absent from the binding protein.

In another embodiment, the invention provides a binding proteincomprising a polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second light chain variable domain obtained from asecond parent antibody or antigen binding portion thereof; C is a lightchain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n does notcomprise an Fc region, wherein said (X2)n is either present or absent.In an embodiment, (X2)n is absent from the binding protein.

In another embodiment the binding protein of the invention comprisesfirst and second polypeptide chains, wherein said first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a firstheavy chain variable domain obtained from a first parent antibody orantigen binding portion thereof; VD2 is a second heavy chain variabledomain obtained from a second parent antibody or antigen binding portionthereof; C is a heavy chain constant domain; (X1)n is a linker with theproviso that it is not CH1, wherein said (X1)n is either present orabsent; and (X2)n is an Fc region, wherein said (X2)n is either presentor absent; and wherein said second polypeptide chain comprises a secondVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second light chain variable domain obtained from asecond parent antibody or antigen binding portion thereof; C is a lightchain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n does notcomprise an Fc region, wherein said (X2)n is either present or absent.In another embodiment, the binding protein comprises two firstpolypeptide chains and two second polypeptide chains. In yet anotherembodiment, (X2)n is absent from the second polypeptide. In stillanother embodiment, the Fc region, if present in the first polypeptideis selected from the group consisting of native sequence Fc region and avariant sequence Fc region. In still another embodiment, the Fc regionis selected from the group consisting of an Fc region from an IgG1,IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In another embodiment the binding protein of the invention is a DVD-Igcapable of binding two antigens comprising four polypeptide chains,wherein, first and third polypeptide chains compriseVD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second heavy chain variable domain obtained from asecond parent antibody or antigen binding portion thereof; C is a heavychain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n is an Fcregion, wherein said (X2)n is either present or absent; and whereinsecond and fourth polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first light chain variable domain obtained from a firstparent antibody or antigen binding portion thereof; VD2 is a secondlight chain variable domain obtained from a second parent antibody orantigen binding portion thereof; C is a light chain constant domain;(X1)n is a linker with the proviso that it is not CH1, wherein said(X1)n is either present or absent; and (X2)n does not comprise an Fcregion, wherein said (X2)n is either present or absent.

The invention provides a method of making a DVD-Ig binding protein bypreselecting the parent antibodies. In an embodiment, the method ofmaking a Dual Variable Domain Immunoglobulin capable of binding twoantigens comprising the steps of a) obtaining a first parent antibody orantigen binding portion thereof, capable of binding a first antigen; b)obtaining a second parent antibody or antigen binding portion thereof,capable of binding a second antigen; c) constructing first and thirdpolypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is afirst heavy chain variable domain obtained from said first parentantibody or antigen binding portion thereof; VD2 is a second heavy chainvariable domain obtained from said second parent antibody or antigenbinding portion thereof; C is a heavy chain constant domain; (X1)n is alinker with the proviso that it is not CH1, wherein said (X1)n is eitherpresent or absent; and (X2)n is an Fc region, wherein said (X2)n iseither present or absent; d) constructing second and fourth polypeptidechains comprising VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first lightchain variable domain obtained from said first parent antibody orantigen binding portion thereof; VD2 is a second light chain variabledomain obtained from said second parent antibody or antigen bindingthereof; C is a light chain constant domain; (X1)n is a linker with theproviso that it is not CH1, wherein said (X1)n is either present orabsent; and (X2)n does not comprise an Fc region, wherein said (X2)n iseither present or absent; e) expressing said first, second, third andfourth polypeptide chains; such that a Dual Variable DomainImmunoglobulin capable of binding said first and said second antigen isgenerated.

In still another embodiment, the invention provides a method ofgenerating a Dual Variable Domain Immunoglobulin capable of binding twoantigens with desired properties comprising the steps of a) obtaining afirst parent antibody or antigen binding portion thereof, capable ofbinding a first antigen and possessing at least one desired propertyexhibited by the Dual Variable Domain Immunoglobulin; b) obtaining asecond parent antibody or antigen binding portion thereof, capable ofbinding a second antigen and possessing at least one desired propertyexhibited by the Dual Variable Domain Immunoglobulin; c) constructingfirst and third polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n,wherein; VD1 is a first heavy chain variable domain obtained from saidfirst parent antibody or antigen binding portion thereof; VD2 is asecond heavy chain variable domain obtained from said second parentantibody or antigen binding portion thereof; C is a heavy chain constantdomain; (X1)n is a linker with the proviso that it is not CH1, whereinsaid (X1)n is either present or absent; and (X2)n is an Fc region,wherein said (X2)n is either present or absent; d) constructing secondand fourth polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein;VD1 is a first light chain variable domain obtained from said firstparent antibody or antigen binding portion thereof; VD2 is a secondlight chain variable domain obtained from said second parent antibody orantigen binding portion thereof; C is a light chain constant domain;(X1)n is a linker with the proviso that it is not CH1, wherein said(X1)n is either present or absent; and (X2)n does not comprise an Fcregion, wherein said (X2)n is either present or absent; e) expressingsaid first, second, third and fourth polypeptide chains; such that aDual Variable Domain Immunoglobulin capable of binding said first andsaid second antigen with desired properties is generated.

In one embodiment, the VDI of the first and second polypeptide chainsdisclosed herein are obtained from the same parent antibody or antigenbinding portion thereof. In another embodiment, the VDI of the first andsecond polypeptide chains disclosed herein are obtained from differentparent antibodies or antigen binding portions thereof. In anotherembodiment, the VD2 of the first and second polypeptide chains disclosedherein are obtained from the same parent antibody or antigen bindingportion thereof. In another embodiment, the VD2 of the first and secondpolypeptide chains disclosed herein are obtained from different parentantibodies or antigen binding portions thereof.

In one embodiment the first parent antibody or antigen binding portionthereof, and the second parent antibody or antigen binding portionthereof, are the same antibody. In another embodiment the first parentantibody or antigen binding portion thereof, and the second parentantibody or antigen binding portion thereof, are different antibodies.

In one embodiment the first parent antibody or antigen binding portionthereof, binds a first antigen and the second parent antibody or antigenbinding portion thereof, binds a second antigen. In a particularembodiment, the first and second antigens are the same antigen. Inanother embodiment, the parent antibodies bind different epitopes on thesame antigen. In another embodiment the first and second antigens aredifferent antigens. In another embodiment, the first parent antibody orantigen binding portion thereof, binds the first antigen with a potencydifferent from the potency with which the second parent antibody orantigen binding portion thereof, binds the second antigen. In yetanother embodiment, the first parent antibody or antigen binding portionthereof, binds the first antigen with an affinity different from theaffinity with which the second parent antibody or antigen bindingportion thereof, binds the second antigen.

In another embodiment the first parent antibody or antigen bindingportion thereof, and the second parent antibody or antigen bindingportion thereof, are selected from the group consisting of, humanantibody, CDR grafted antibody, and humanized antibody. In anembodiment, the antigen binding portions are selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a Fd fragment consisting of the VH and CH1 domains; a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, a dAb fragment, an isolated complementarity determining region(CDR), a single chain antibody, and diabodies.

In another embodiment the binding protein of the invention possesses atleast one desired property exhibited by the first parent antibody orantigen binding portion thereof, or the second parent antibody orantigen binding portion thereof. Alternatively, the first parentantibody or antigen binding portion thereof and the second parentantibody or antigen binding portion thereof possess at least one desiredproperty exhibited by the Dual Variable Domain Immunoglobulin. In anembodiment, the desired property is selected from one or more antibodyparameters. In another embodiment, the antibody parameters are selectedfrom the group consisting of antigen specificity, affinity to antigen,potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, and orthologous antigenbinding. In an embodiment the binding protein is multivalent. In anotherembodiment, the binding protein is multispecific. The multivalent and ormultispecific binding proteins described herein have desirableproperties particularly from a therapeutic standpoint. For instance, themultivalent and or multispecific binding protein may (1) be internalized(and/or catabolized) faster than a bivalent antibody by a cellexpressing an antigen to which the antibodies bind; (2) be an agonistantibody; and/or (3) induce cell death and/or apoptosis of a cellexpressing an antigen which the multivalent antibody is capable ofbinding to. The “parent antibody” which provides at least one antigenbinding specificity of the multivalent and or multispecific bindingproteins may be one which is internalized (and/or catabolized) by a cellexpressing an antigen to which the antibody binds; and/or may be anagonist, cell death-inducing, and/or apoptosis-inducing antibody, andthe multivalent and or multispecific binding protein as described hereinmay display improvement(s) in one or more of these properties. Moreover,the parent antibody may lack any one or more of these properties, butmay be endowed with them when constructed as a multivalent bindingprotein as described herein.

In another embodiment the binding protein of the invention has an onrate constant (Kon) to one or more targets selected from the groupconsisting of: at least about 10²M⁻¹ s⁻¹; at least about 10³M⁻¹ s⁻¹; atleast about 10⁴M⁻¹ s⁻¹; at least about 10⁵M⁻¹ s⁻¹; and at least about10⁶M⁻¹ s⁻¹, as measured by surface plasmon resonance. In an embodiment,the binding protein of the invention has an on rate constant (Kon) toone or more targets between 10²M⁻¹ s⁻¹ and 10³M⁻¹ s⁻¹; between 10³M⁻¹s⁻¹ and 10⁴M⁻¹ s⁻¹; between 10⁴M⁻¹ s⁻¹ and 10⁵M⁻¹ s⁻¹; or between 10⁵M⁻¹s⁻¹ and 106M⁻¹ s⁻¹, as measured by surface plasmon resonance.

In another embodiment the binding protein has an off rate constant(Koff) for one or more targets selected from the group consisting of: atmost about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; andat most about 10⁻⁶ s⁻¹, as measured by surface plasmon resonance. In anembodiment, the binding protein of the invention has an off rateconstant (Koff) to one or more targets of 10⁻³ s⁻¹ to 10⁻⁴ s⁻¹; of 10⁻⁴s⁻¹ to 10⁻⁵ s⁻¹; or of 10⁻⁵ s⁻¹ to 10⁻⁶ s¹, as measured by surfaceplasmon resonance.

In another embodiment the binding protein has a dissociation constant(K_(D)) to one or more targets selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³M. In an embodiment, the binding protein of the invention has adissociation constant (K_(D)) to its targets of 10⁻⁷ M to 10⁻⁸ M; of10⁻⁸ M to 10⁻⁹ M; of 10⁻¹⁰ M to 10⁻¹⁰ M; of 10⁻¹⁰ to 10⁻¹¹ M; of 10⁻¹¹ Mto 10⁻¹² M; m or of 10⁻¹² to M 10⁻¹³ M.

In another embodiment, the binding protein described herein is aconjugate further comprising an agent selected from the group consistingof an immunoadhesion molecule, an imaging agent, a therapeutic agent,and a cytotoxic agent. In an embodiment, the imaging agent is selectedfrom the group consisting of a radiolabel, an enzyme, a fluorescentlabel, a luminescent label, a bioluminescent label, a magnetic label,and biotin. In another embodiment, the imaging agent is a radiolabelselected from the group consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In yet another embodiment, thetherapeutic or cytotoxic agent is selected from the group consisting ofan anti-metabolite, an alkylating agent, an antibiotic, a growth factor,a cytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, toxin, and an apoptotic agent.

In another embodiment, the binding protein described herein is acrystallized binding protein and exists as a crystal. In an embodiment,the crystal is a carrier-free pharmaceutical controlled release crystal.In yet another embodiment, the crystallized binding protein has agreater half life in vivo than the soluble counterpart of said bindingprotein. In still another embodiment, the crystallized binding proteinretains biological activity.

In another embodiment, the binding protein described herein isglycosylated. For example, the glycosylation is a human glycosylationpattern.

One aspect of the invention pertains to an isolated nucleic acidencoding any one of the binding proteins disclosed herein. A furtherembodiment provides a vector comprising the isolated nucleic aciddisclosed herein wherein said vector is selected from the groupconsisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002,Vol 30, No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS(Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No.17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. In an embodiment, thevector is a vector disclosed in U.S. Patent Application Ser. No.61/021,282.

In another aspect a host cell is transformed with the vector disclosedherein. In an embodiment, the host cell is a prokaryotic cell. Inanother embodiment, the host cell is E. Coli. In a related embodimentthe host cell is a eukaryotic cell. In another embodiment, theeukaryotic cell is selected from the group consisting of protist cell,animal cell, plant cell and fungal cell. In yet another embodiment, thehost cell is a mammalian cell including, but not limited to, CHO, COS;NS0, SP2, PER.C6 or a fungal cell such as Saccharomyces cerevisiae; oran insect cell such as Sf9.

In an embodiment, two or more DVD-Igs, e.g., with differentspecificities, are produced in a single recombinant host cell. Forexample, the expression of a mixture of antibodies has been calledOligoclonics™, (Merus B.V., The Netherlands) U.S. Pat. Nos. 7,262,028;7,429,486.

Another aspect of the invention provides a method of producing a bindingprotein disclosed herein comprising culturing any one of the host cellsalso disclosed herein in a culture medium under conditions sufficient toproduce the binding protein. In an embodiment, 50%-75% of the bindingprotein produced by this method is a dual specific tetravalent bindingprotein. In a particular embodiment, 75%-90% of the binding proteinproduced by this method is a dual specific tetravalent binding protein.In a particular embodiment, 90%-95% of the binding protein produced is adual specific tetravalent binding protein.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a formulation that in turncomprises a crystallized binding protein, as disclosed herein, and aningredient, and at least one polymeric carrier. For example, thepolymeric carrier is a polymer selected from one or more of the groupconsisting of: poly (acrylic acid), poly (cyanoacrylates), poly (aminoacids), poly (anhydrides), poly (depsipeptide), poly (esters), poly(lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly(ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof. For example, the ingredient is selected from thegroup consisting of albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol. Another embodiment provides a method for treating amammal comprising the step of administering to the mammal an effectiveamount of the composition disclosed herein.

The invention also provides a pharmaceutical composition comprising abinding protein, as disclosed herein and a pharmaceutically acceptablecarrier. In a further embodiment the pharmaceutical compositioncomprises at least one additional therapeutic agent for treating adisorder. For example, the additional agent is selected from the groupconsisting of: a therapeutic agent, an imaging agent, a cytotoxic agent,an angiogenesis inhibitor (including but not limited to an anti-VEGFantibody or a VEGF-trap), a kinase inhibitor (including but not limitedto a KDR and a TIE-2 inhibitor), a co-stimulation molecule blocker(including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig,anti-CD20), an adhesion molecule blocker (including but not limited toan anti-LFA-1 antibody, an anti-E/L selectin antibody, a small moleculeinhibitor), an anti-cytokine antibody or functional fragment thereof(including but not limited to an anti-IL-18, an anti-TNF, and ananti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin,rapamycin, FK506, a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,and a cytokine antagonist.

In another aspect, the invention provides a method for treating a humansubject suffering from a disorder in which the target, or targets,capable of being bound by the binding protein disclosed herein isdetrimental, comprising administering to the human subject a bindingprotein disclosed herein such that the activity of the target, ortargets in the human subject is inhibited and one of more symptoms isalleviated or treatment is achieved. For example, the disorder isselected from the group comprising arthritis, osteoarthritis, juvenilechronic arthritis, septic arthritis, Lyme arthritis, psoriaticarthritis, reactive arthritis, spondyloarthropathy, systemic lupuserythematosus, Crohn's disease, ulcerative colitis, inflammatory boweldisease, insulin dependent diabetes mellitus, thyroiditis, asthma,allergic diseases, psoriasis, dermatitis scleroderma, graft versus hostdisease, organ transplant rejection, acute or chronic immune diseaseassociated with organ transplantation, sarcoidosis, atherosclerosis,disseminated intravascular coagulation, Kawasaki's disease, Grave'sdisease, nephrotic syndrome, chronic fatigue syndrome, Wegener'sgranulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis ofthe kidneys, chronic active hepatitis, uveitis, septic shock, toxicshock syndrome, sepsis syndrome, cachexia, infectious diseases,parasitic diseases, acquired immunodeficiency syndrome, acute transversemyelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease,stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadicpolyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia areata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-l-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aortic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chronic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic diseases, migraine headache,mitochondrial multi.system disorder, mixed connective tissue disease,monoclonal gammopathy, multiple myeloma, multiple systems degenerations(Mencel Dej erine-Thomas Shi-Drager and Machado-Joseph), myastheniagravis, mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occlusive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods of the invention include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

The DVD-Igs of the invention may also treat one or more of the followingdiseases: Acute coronary syndromes, Acute Idiopathic Polyneuritis, AcuteInflammatory Demyelinating Polyradiculoneuropathy, Acute ischemia, AdultStill's Disease, Alopecia areata, Anaphylaxis, Anti-PhospholipidAntibody Syndrome, Aplastic anemia, Arteriosclerosis, Atopic eczema,Atopic dermatitis, Autoimmune dermatitis, Autoimmune disorder associatedwith Streptococcus infection, autoimmune enteropathy, Autoimmunehearingloss, Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmunemyocarditis, autoimmune thrombocytopenia (AITP), autoimmune prematureovarian failure, Blepharitis, Bronchiectasis, Bullous pemphigoid,Cardiovascular Disease, Catastrophic Antiphospholipid Syndrome, CeliacDisease, Cervical Spondylosis, Chronic ischemia, Cicatricial pemphigoid,Clinically isolated Syndrome (CIS) with Risk for Multiple Sclerosis,Conjunctivitis, Childhood Onset Psychiatric Disorder, Chronicobstructive pulmonary disease (COPD), Dacryocystitis, dermatomyositis,Diabetic retinopathy, Diabetes mellitus, Disk herniation, Disk prolaps,Drug induced immune hemolytic anemia, Endocarditis, Endometriosis,endophthalmitis, Episcleritis, Erythema multiforme, erythema multiformemajor, Gestational pemphigoid, Guillain-Barré Syndrome (GBS), Hay Fever,Hughes Syndrome, Idiopathic Parkinson's Disease, idiopathic interstitialpneumonia, IgE-mediated Allergy, Immune hemolytic anemia, Inclusion BodyMyositis, Infectious ocular inflammatory disease, Inflammatorydemyelinating disease, Inflammatory heart disease, Inflammatory kidneydisease, IPF/UIP, Iritis, Keratitis, Keratojuntivitis sicca, Kussmauldisease or Kussmaul-Meier Disease, Landry's Paralysis, Langerhan's CellHistiocytosis, Livedo reticularis, Macular Degeneration, malignancies,Microscopic Polyangiitis, Morbus Bechterev, Motor Neuron Disorders,Mucous membrane pemphigoid, Multiple Organ failure, Myasthenia Gravis,Myelodysplastic Syndrome, Myocarditis, Nerve Root Disorders, Neuropathy,Non-A Non-B Hepatitis, Optic Neuritis, Osteolysis, Ovarian cancer,Pauciarticular JRA, peripheral artery occlusive disease (PAOD),peripheral vascular disease (PVD), peripheral artery disease (PAD),Phlebitis, Polyarteritis nodosa (or periarteritis nodosa),Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA,Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica(PMR), Post-Pump Syndrome, primary Parkinsonism, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency,Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease,SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis),Scleroderma, Secondary Amyloidosis, Shock lung, Scleritis, Sciatica,Secondary Adrenal Insufficiency, Silicone associated connective tissuedisease, Sneddon-Wilkinson Dermatosis, spondilitis ankylosans,Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome,Temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1allergic reaction, Type II Diabetes, Urticaria, Usual interstitialpneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular degeneration,Wound healing, yersinia, and salmonella associated arthropathy.

In an embodiment, the antibodies of the invention or antigen-bindingportions thereof, are used to treat cancer or in the prevention ofmetastases from the tumors described herein either when used alone or incombination with radiotherapy and/or other chemotherapeutic agents.

In another aspect the invention provides a method of treating a patientsuffering from a disorder comprising the step of administering any oneof the binding proteins disclosed herein before, concurrent, or afterthe administration of a second agent, as discussed herein. In aparticular embodiment the second agent is selected from the groupconsisting of budenoside, epidermal growth factor, corticosteroids,cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine,azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine,olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1receptor antagonists, anti-IL-1β mAbs, anti-IL-6 or IL-6 receptor mAbs,growth factors, elastase inhibitors, pyridinyl-imidazole compounds,antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12,IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF,antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45,CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506,rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosineagonists, antithrombotic agents, complement inhibitors, adrenergicagents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β convertingenzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signallinginhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor,sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,IL-11, IL-13 and TGFβ. In a particular embodiment the pharmaceuticalcompositions disclosed herein are administered to the patient by atleast one mode selected from parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

One aspect of the invention provides at least one anti-idiotype antibodyto at least one binding protein of the present invention. Theanti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, or any portion thereof,that can be incorporated into a binding protein of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of Dual Variable Domain (DVD)-Igconstructs and shows the strategy for generation of a DVD-Ig from twoparent antibodies;

FIG. 1B, is a schematic representation of constructs DVD1-Ig, DVD2-Ig,and two chimeric mono-specific antibodies from hybridoma clones 2D13.E3(anti-IL-1α) and 13F5.G5 (anti-IL-1β).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to multivalent and/or multispecific bindingproteins capable of binding two or more antigens. Specifically, theinvention relates to dual variable domain immunoglobulins (DVD-Ig), andpharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such DVD-Igs.Methods of using the DVD-Igs of the invention to detect specificantigens, either in vitro or in vivo are also encompassed by theinvention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric. Use of “polypeptide” hereinis intended to encompass polypeptide and fragments and variants(including fragments of variants) thereof, unless otherwise contradictedby context. For an antigenic polypeptide, a fragment of polypeptideoptionally contains at least one contiguous or nonlinear epitope ofpolypeptide. The precise boundaries of the at least one epitope fragmentcan be confirmed using ordinary skill in the art. The fragment comprisesat least about 5 contiguous amino acids, such as at least about 10contiguous amino acids, at least about 15 contiguous amino acids, or atleast about 20 contiguous amino acids. A variant of polypeptide is asdescribed herein.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

“Biological activity” as used herein, refers to any one or more inherentbiological properties of a molecule (whether present naturally as foundin vivo, or provided or enabled by recombinant means). Biologicalproperties include but are not limited to binding receptor; induction ofcell proliferation, inhibiting cell growth, inductions of othercytokines, induction of apoptosis, and enzymatic activity. Biologicalactivity also includes activity of an Ig molecule.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (Winter, et al. U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fcportion of an antibody mediates several important effector functionse.g., cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered. The dimerization oftwo identical heavy chains of an immunoglobulin is mediated by thedimerization of CH3 domains and is stabilized by the disulfide bondswithin the hinge region (Huber et al. Nature; 264: 415-20; Thies et al1999 J Mol Biol; 293: 67-79.). Mutation of cysteine residues within thehinge regions to prevent heavy chain-heavy chain disulfide bonds willdestabilize dimeration of CH3 domains. Residues responsible for CH3dimerization have been identified (Dall'Acqua 1998 Biochemistry 37:9266-73.). Therefore, it is possible to generate a monovalent half-Ig.Interestingly, these monovalent half Ig molecules have been found innature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129:855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). Thestoichiometry of FcRn: Ig Fc region has been determined to be 2:1 (Westet al 0.2000 Biochemistry 39: 9698-708), and half Fc is sufficient formediating FcRn binding (Kim et al 1994 Eur J Immunol; 24: 542-548.).Mutations to disrupt the dimerization of CH3 domain may not have greateradverse effect on its FcRn binding as the residues important for CH3dimerization are located on the inner interface of CH3 b sheetstructure, whereas the region responsible for FcRn binding is located onthe outside interface of CH2-CH3 domains. However the half Ig moleculemay have certain advantage in tissue penetration due to its smaller sizethan that of a regular antibody. In one embodiment at least one aminoacid residue is replaced in the constant region of the binding proteinof the invention, for example the Fc region, such that the dimerizationof the heavy chains is disrupted, resulting in half DVD Ig molecules.The anti-inflammatory activity of IgG is dependent on sialylation of theN-linked glycan of the IgG Fc fragment. The precise glycan requirementsfor anti-inflammatory activity has been determined, such that anappropriate IgG1 Fc fragment can be created, thereby generating a fullyrecombinant, sialylated IgG1 Fc with greatly enhanced potency (Anthony,R. M., et al. (2008) Science 320:373-376).

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5). In addition single chain antibodies also include“linear antibodies” comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

The term “multivalent binding protein” is used throughout thisspecification to denote a binding protein comprising two or more antigenbinding sites. In an embodiment, the multivalent binding protein isengineered to have the three or more antigen binding sites, and isgenerally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein capable of binding two ormore related or unrelated targets. Dual variable domain (DVD) bindingproteins of the invention comprise two or more antigen binding sites andare tetravalent or multivalent binding proteins. DVDs may bemonospecific, i.e., capable of binding one antigen or multispecific,i.e. capable of binding two or more antigens. DVD binding proteinscomprising two heavy chain DVD polypeptides and two light chain DVDpolypeptides are referred to as DVD-Ig. Each half of a DVD-Ig comprisesa heavy chain DVD polypeptide, and a light chain DVD polypeptide, andtwo antigen binding sites. Each binding site comprises a heavy chainvariable domain and a light chain variable domain with a total of 6 CDRsinvolved in antigen binding per antigen binding site.

The term “bispecific antibody”, as used herein, refers to full-lengthantibodies that are generated by quadroma technology (see Milstein, C.and A.C. Cuello, Nature, 1983. 305(5934): p. 537-40), by chemicalconjugation of two different monoclonal antibodies (see Staerz, U. D.,et al., Nature, 1985. 314(6012): p. 628-31), or by knob-into-hole orsimilar approaches which introduces mutations in the Fc region (seeHolliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993.90(14): p. 6444-8.18), resulting in multiple different immunoglobulinspecies of which only one is the functional bispecific antibody. Bymolecular function, a bispecific antibody binds one antigen (or epitope)on one of its two binding arms (one pair of HC/LC), and binds adifferent antigen (or epitope) on its second arm (a different pair ofHC/LC). By this definition, a bispecific antibody has two distinctantigen binding arms (in both specificity and CDR sequences), and ismonovalent for each antigen it binds to.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen it binds to.

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

The term “cytokine” is a generic term for proteins released by one cellpopulation, which act on another cell population as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-alpha;platelet-growth factor; placental growth factor, transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-1 and -11; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, -beta and -gamma colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-15, IL-18, IL-21, IL-22, IL-23, IL-33; a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

The term “linker” is used to denote polypeptides comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Exemplary linkers include, but are not limited to, AKTTPKLEEGEFSEAR (SEQID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6);RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄(SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO:11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP(SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16);AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ IDNO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); andGHEAAAVMQVQYPAS (SEQ ID NO: 26).

An “immunoglobulin constant domain” refers to a heavy or light chainconstant domain. Human IgG heavy chain and light chain constant domainamino acid sequences are known in the art.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), each mAbis directed against a single determinant on the antigen. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II C, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom H. R. (1997) TIB Tech.15:62-70; Azzazy H., and Highsmith W.E. (2002) Clin. Biochem.35:425-445; Gavilondo J.V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see, Taylor, L. D., et al.(1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A. and Green L. L.(2002) Current Opinion in Biotechnology 13:593-597; Little M. et al.(2000) Immunology Today 21:364-370) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Exemplary affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. BidlTechnology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al, J. Mol.BioL 226:889-896 (1992) and selective mutation at selective mutagenesispositions, contact or hypermutation positions with an activity enhancingamino acid residue as described in U.S. Pat. No. 6,914,128B1.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. Also “humanized antibody” is an antibody or a variant,derivative, analog or fragment thereof which immunospecifically binds toan antigen of interest and which comprises a framework (FR) regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. In an embodiment, a humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. In someembodiments, a humanized antibody contains both the light chain as wellas at least the variable domain of a heavy chain. The antibody also mayinclude the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Insome embodiments, a humanized antibody only contains a humanized lightchain. In some embodiments, a humanized antibody only contains ahumanized heavy chain. In specific embodiments, a humanized antibodyonly contains a humanized variable domain of a light chain and/orhumanized heavy chain.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the herein systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although certain embodiments useKabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the recognition that germline antibody genes aremore likely than mature antibody genes to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as from a foreign source when usedtherapeutically in that species.

As used herein, the term “neutralizing” refers to counteracting thebiological activity of an antigen when a binding protein specificallybinds the antigen. In an embodiment, the neutralizing binding proteinbinds the cytokine and reduces its biologically activity by at leastabout 20%, 40%, 60%, 80%, 85% or more.

The term “activity” includes activities such as the binding specificityand affinity of a DVD-Ig for two or more antigens.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it recognizes its target antigen in a complexmixture of proteins and/or macromolecules. Antibodies are said to “bindto the same epitope” if the antibodies cross-compete (one prevents thebinding or modulating effect of the other). In addition structuraldefinitions of epitopes (overlapping, similar, identical) areinformative, but functional definitions are often more relevant as theyencompass structural (binding) and functional (modulation, competition)parameters.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore® system(BIAcore International AB, a GE Healthcare company, Uppsala, Sweden andPiscataway, N.J.). For further descriptions, see Jönsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of a binding protein (e.g., an antibody) to theantigen to form the, e.g., antibody/antigen complex as is known in theart. The “Kon” also is known by the terms “association rate constant”,or “ka”, as used interchangeably herein. This value indicating thebinding rate of an antibody to its target antigen or the rate of complexformation between an antibody and antigen also is shown by the equationbelow:

Antibody(“Ab”)+Antigen(“Ag”)→Ab−Ag.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation, or “dissociation rate constant”, of a bindingprotein (e.g., an antibody) from the, e.g., antibody/antigen complex asis known in the art. This value indicates the dissociation rate of anantibody from its target antigen or separation of Ab-Ag complex overtime into free antibody and antigen as shown by the equation below:

Ab+Ag→Ab−Ag.

The term “K_(D)”, as used herein, is intended to refer to the“equilibrium dissociation constant”, and refers to the value obtained ina titration measurement at equilibrium, or by dividing the dissociationrate constant (koff) by the association rate constant (kon). Theassociation rate constant, the dissociation rate constant and theequilibrium dissociation constant are used to represent the bindingaffinity of an antibody to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore®(biomolecular interaction analysis) assay can be used (e.g., instrumentavailable from BIAcore International AB, a GE Healthcare company,Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay)assay, available from Sapidyne Instruments (Boise, Id.) can also beused.

“Label” and “detectable label” mean a moiety attached to a specificbinding partner, such as an antibody or an analyte, e.g., to render thereaction between members of a specific binding pair, such as an antibodyand an analyte, detectable, and the specific binding partner, e.g.,antibody or analyte, so labeled is referred to as “detectably labeled.”Thus, the term “labeled binding protein” as used herein, refers to aprotein with a label incorporated that provides for the identificationof the binding protein. In an embodiment, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Examples of labels for polypeptides include, but are notlimited to, the following: radioisotopes or radionuclides (e.g., ³H ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); chromogens,fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, luciferase, alkalinephosphatase); chemiluminescent markers; biotinyl groups; predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are described herein. In this regard, the moiety itself maynot be detectably labeled but may become detectable upon reaction withyet another moiety. Use of “detectably labeled” is intended to encompassthe latter type of detectable labeling.

The term “conjugate” refers to a binding protein, such as an antibody,chemically linked to a second chemical moiety, such as a therapeutic orcytotoxic agent. The term “agent” is used herein to denote a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. In an embodiment, thetherapeutic or cytotoxic agents include, but are not limited to,pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, the conjugate antibody may be a detectably labeledantibody used as the detection antibody.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein (e.g., an antibody), or antigen binding portion thereof,that exists in the form of a crystal. Crystals are one form of the solidstate of matter, which is distinct from other forms such as theamorphous solid state or the liquid crystalline state. Crystals arecomposed of regular, repeating, three-dimensional arrays of atoms, ions,molecules (e.g., proteins such as antibodies), or molecular assemblies(e.g., antigen/antibody complexes). These three-dimensional arrays arearranged according to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, Crystallization of Nucleic Acids and Proteins, a PracticalApproach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y.,(1999).”

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The term includes single and doublestranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide” is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector”, is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, refers to any process by which exogenous DNA enters ahost cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the host cell being transformed and may include, butis not limited to, viral infection, electroporation, lipofection, andparticle bombardment. Such “transformed” cells include stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome. They also include cells which transiently express theinserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended torefer to a cell into which exogenous DNA has been introduced. In anembodiment, the host cell comprises two more more (e.g., multiple)nucleic acids encoding antibodies, such as the host cells described inU.S. Pat. No. 7,262,028, for example. It should be understood that suchterms are intended to refer not only to the particular subject cell, butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. In an embodiment, host cells include prokaryotic andeukaryotic cells selected from any of the Kingdoms of life. In anotherembodiment, eukaryotic cells include protist, fungal, plant and animalcells. In another embodiment, host cells include but are not limited tothe prokaryotic cell line E. Coli; mammalian cell lines CHO, HEK 293,COS, NS0, SP2 and PER.C6; the insect cell line Sf9; and the fungal cellSaccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art, refers to an organism havingcells that contain a transgene, wherein the transgene introduced intothe organism (or an ancestor of the organism) expresses a polypeptidenot naturally expressed in the organism. A “transgene” is a DNAconstruct, which is stably and operably integrated into the genome of acell from which a transgenic organism develops, directing the expressionof an encoded gene product in one or more cell types or tissues of thetransgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of a cytokine).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator” is a compound capable of changingor altering an activity or function of a molecule of interest (e.g., thebiological activity of a cytokine). For example, a modulator may causean increase or decrease in the magnitude of a certain activity orfunction of a molecule compared to the magnitude of the activity orfunction observed in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Exemplary inhibitorsinclude, but are not limited to, proteins, peptides, antibodies,peptibodies, carbohydrates or small organic molecules. Peptibodies aredescribed, e.g., in WO01/83525.

The term “agonist”, refers to a modulator that, when contacted with amolecule of interest, causes an increase in the magnitude of a certainactivity or function of the molecule compared to the magnitude of theactivity or function observed in the absence of the agonist. Particularagonists of interest may include, but are not limited to, polypeptides,nucleic acids, carbohydrates, or any other molecules that bind to theantigen.

The term “antagonist” or “inhibitor”, refer to a modulator that, whencontacted with a molecule of interest causes a decrease in the magnitudeof a certain activity or function of the molecule compared to themagnitude of the activity or function observed in the absence of theantagonist. Particular antagonists of interest include those that blockor modulate the biological or immunological activity of of the antigen.Antagonists and inhibitors of antigens may include, but are not limitedto, proteins, nucleic acids, carbohydrates, or any other molecules,which bind to the antigen.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a mammal, including a primate (for example, a human,a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, a mouse, a whale), a bird (e.g., a duck or agoose), and a shark. Preferably, the patient or subject is a human, suchas a human being treated or assessed for a disease, disorder orcondition, a human at risk for a disease, disorder or condition, a humanhaving a disease, disorder or condition, and/or human being treated fora disease, disorder or condition.

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood), plasma, serum,urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

“Component,” “components,” and “at least one component,” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure, “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negativecontrol”) or to contain analyte (“positive control”). A positive controlcan comprise a known concentration of analyte. “Control,” “positivecontrol,” and “calibrator” may be used interchangeably herein to referto a composition comprising a known concentration of analyte. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) should be generallyapplicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte (e.g.,polypeptide of interest) may entail release of the analyte from anyendogenous binding proteins present in the sample. A pretreatmentreagent may be homogeneous (not requiring a separation step) orheterogeneous (requiring a separation step). With use of a heterogeneouspretreatment reagent there is removal of any precipitated analytebinding proteins from the test sample prior to proceeding to the nextstep of the assay.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen (or fragmentthereof) and an antibody (or antigenically reactive fragment thereof))refer to the selective reactivity of the interaction. The phrase“specifically binds to” and analogous phrases refer to the ability ofantibodies (or antigenically reactive fragments thereof) to bindspecifically to analyte (or a fragment thereof) and not bindspecifically to other entities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes, fragments, and variants (includingfragments of variants) thereof, whether isolated or recombinantlyproduced.

“Variant” as used herein means a polypeptide that differs from a givenpolypeptide (e.g., IL-18, BNP, NGAL or HIV polypeptide oranti-polypeptide antibody) in amino acid sequence by the addition (e.g.,insertion), deletion, or conservative substitution of amino acids, butthat retains the biological activity of the given polypeptide (e.g., avariant IL-18 can compete with anti-IL-18 antibody for binding toIL-18). A conservative substitution of an amino acid, i.e., replacing anamino acid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes off 2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101, which is incorporated herein by reference). Substitution ofamino acids having similar hydrophilicity values can result in peptidesretaining biological activity, for example immunogenicity, as isunderstood in the art. In one aspect, substitutions are performed withamino acids having hydrophilicity values within ±2 of each other. Boththe hydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. “Variant” also can be used to describe apolypeptide or fragment thereof that has been differentially processed,such as by proteolysis, phosphorylation, or other post-translationalmodification, yet retains its biological activity or antigen reactivity,e.g., the ability to bind to IL-18. Use of “variant” herein is intendedto encompass fragments of a variant unless otherwise contradicted bycontext.

I. Generation of DVD Binding Protein

The invention pertains to Dual Variable Domain binding proteins capableof binding one or more targets and methods of making the same. In anembodiment, the binding protein comprises a polypeptide chain, whereinsaid polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is afirst variable domain, VD2 is a second variable domain, C is a constantdomain, X1 represents an amino acid or polypeptide, X2 represents an Fcregion and n is 0 or 1. The binding protein of the invention can begenerated using various techniques. The invention provides expressionvectors, host cell and methods of generating the binding protein.

A. Generation of Parent Monoclonal Antibodies

The variable domains of the DVD binding protein can be obtained fromparent antibodies, including polyclonal and mAbs capable of bindingantigens of interest. These antibodies may be naturally occurring or maybe generated by recombinant technology.

MAbs can be prepared using a wide variety of techniques known in the artincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, mAbs can beproduced using hybridoma techniques including those known in the art andtaught, for example, in Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. Hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed in Example 1 below. Hybridomas may becultured and expanded in vivo in syngeneic animals, in animals that lackan immune system, e.g., nude mice, or in cell culture in vitro. Methodsof selecting, cloning and expanding hybridomas are well known to thoseof ordinary skill in the art. In a particular embodiment, the hybridomasare mouse hybridomas. In another embodiment, the hybridomas are producedin a non-human, non-mouse species such as rats, sheep, pigs, goats,cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an antibody capable of binding a specific antigen.

Recombinant mAbs are also generated from single, isolated lymphocytesusing a procedure referred to in the art as the selected lymphocyteantibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCTPublication WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl.Acad. Sci. USA 93:7843-7848. In this method, single cells secretingantibodies of interest, e.g., lymphocytes derived from an immunizedanimal, are identified, and, heavy- and light-chain variable regioncDNAs are rescued from the cells by reverse transcriptase-PCR and thesevariable regions can then be expressed, in the context of appropriateimmunoglobulin constant regions (e.g., human constant regions), inmammalian host cells, such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences, derived from invivo selected lymphocytes, can then undergo further analysis andselection in vitro, for example by panning the transfected cells toisolate cells expressing antibodies to the antigen of interest. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

Monoclonal antibodies are also produced by immunizing a non-human animalcomprising some, or all, of the human immunoglobulin locus with anantigen of interest. In an embodiment, the non-human animal is aXENOMOUSE transgenic mouse, an engineered mouse strain that compriseslarge fragments of the human immunoglobulin loci and is deficient inmouse antibody production. See, e.g., Green et al. Nature Genetics7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615,5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO91/10741, published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994,WO 96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654,published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999,WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24,2000 and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE transgenicmouse produces an adult-like human repertoire of fully human antibodies,and generates antigen-specific human monoclonal antibodies. TheXENOMOUSE transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of whichare hereby incorporated by reference.

In vitro methods also can be used to make the parent antibodies, whereinan antibody library is screened to identify an antibody having thedesired binding specificity. Methods for such screening of recombinantantibody libraries are well known in the art and include methodsdescribed in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kanget al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No.WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland etal. PCT Publication No. WO 92/15679; Breitling et al. PCT PublicationNo. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al.,Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J12:725-734;Hawkins et al. (1992) J Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patentapplication publication 20030186374, and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

Parent antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e. g., human or murine). Phage expressing an antigen binding domainthat binds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the herein references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258, 498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of parentantibodies. One type of alternative expression system is one in whichthe recombinant antibody library is expressed as RNA-protein fusions, asdescribed in PCT Publication No. WO 98/31700 by Szostak and Roberts, andin Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, a covalent fusion is created between anmRNA and the peptide or protein that it encodes by in vitro translationof synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic,at their 3′ end. Thus, a specific mRNA can be enriched from a complexmixture of mRNAs (e.g., a combinatorial library) based on the propertiesof the encoded peptide or protein, e.g., antibody, or portion thereof,such as binding of the antibody, or portion thereof, to the dualspecificity antigen. Nucleic acid sequences encoding antibodies, orportions thereof, recovered from screening of such libraries can beexpressed by recombinant means as described herein (e.g., in mammalianhost cells) and, moreover, can be subjected to further affinitymaturation by either additional rounds of screening of mRNA-peptidefusions in which mutations have been introduced into the originallyselected sequence(s), or by other methods for affinity maturation invitro of recombinant antibodies, as described herein.

In another approach the parent antibodies can also be generated usingyeast display methods known in the art. In yeast display methods,genetic methods are used to tether antibody domains to the yeast cellwall and display them on the surface of yeast. In particular, such yeastcan be utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Examples of yeast display methods that can be used to make the parentantibodies include those disclosed in Wittrup, et al. U.S. Pat. No.6,699,658 incorporated herein by reference.

The antibodies described herein can be further modified to generate CDRgrafted and humanized parent antibodies. CDR-grafted parent antibodiescomprise heavy and light chain variable region sequences from a humanantibody wherein one or more of the CDR regions of V_(H) and/or V_(L)are replaced with CDR sequences of murine antibodies capable of bindingantigen of interest. A framework sequence from any human antibody mayserve as the template for CDR grafting. However, straight chainreplacement onto such a framework often leads to some loss of bindingaffinity to the antigen. The more homologous a human antibody is to theoriginal murine antibody, the less likely the possibility that combiningthe murine CDRs with the human framework will introduce distortions inthe CDRs that could reduce affinity. Therefore, in an embodiment, thehuman variable framework that is chosen to replace the murine variableframework apart from the CDRs have at least a 65% sequence identity withthe murine antibody variable region framework. In an embodiment, thehuman and murine variable regions apart from the CDRs have at least 70%sequence identify. In a particular embodiment, that the human and murinevariable regions apart from the CDRs have at least 75% sequenceidentity. In another embodiment, the human and murine variable regionsapart from the CDRs have at least 80% sequence identity. Methods forproducing such antibodies are known in the art (see EP 239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., ProteinEngineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973(1994)), and chain shuffling (U.S. Pat. No. 5,565,352); andanti-idiotypic antibodies.

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;www.ibt.unam.mx/virNmice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;www.ibt.unam.mx/vinstructure/stataim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.about.finolina/Web-pages/Pept/spottech.html;www.jerini.de/frroducts.htm; www.patents.ibm.com/ibm.html. Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,e.g., improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

B. Criteria for Selecting Parent Monoclonal Antibodies

An embodiment of the invention pertains to selecting parent antibodieswith at least one or more properties desired in the DVD-Ig molecule. Inan embodiment, the desired property is selected from one or moreantibody parameters. In another embodiment, the antibody parameters areselected from the group consisting of antigen specificity, affinity toantigen, potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, and orthologous antigenbinding.

B1. Affinity to Antigen

The desired affinity of a therapeutic mAb may depend upon the nature ofthe antigen, and the desired therapeutic end-point. In an embodiment,monoclonal antibodies have higher affinities (Kd=0.01-0.50 pM) whenblocking a cytokine-cytokine receptor interaction as such interactionare usually high affinity interactions (e.g., <pM-<nM ranges). In suchinstances, the mAb affinity for its target should be equal to or betterthan the affinity of the cytokine (ligand) for its receptor. On theother hand, mAb with lesser affinity (>nM range) could betherapeutically effective e.g., in clearing circulating potentiallypathogenic proteins e.g., monoclonal antibodies that bind to, sequester,and clear circulating species of A-β amyloid. In other instances,reducing the affinity of an existing high affinity mAb by site-directedmutagenesis or using a mAb with lower affinity for its target could beused to avoid potential side-effects e.g., a high affinity mAb maysequester/neutralize all of its intended target, thereby completelydepleting/eliminating the function(s) of the targeted protein. In thisscenario, a low affinity mAb may sequester/neutralize a fraction of thetarget that may be responsible for the disease symptoms (thepathological or over-produced levels), thus allowing a fraction of thetarget to continue to perform its normal physiological function(s).Therefore, it may be possible to reduce the Kd to adjust dose and/orreduce side-effects. The affinity of the parental mAb might play a rolein appropriately targeting cell surface molecules to achieve desiredtherapeutic out-come. For example, if a target is expressed on cancercells with high density and on normal cells with low density, a loweraffinity mAb will bind a greater number of targets on tumor cells thannormal cells, resulting in tumor cell elimination via ADCC or CDC, andtherefore might have therapeutically desirable effects. Thus selecting amAb with desired affinity may be relevant for both soluble and surfacetargets.

Signaling through a receptor upon interaction with its ligand may dependupon the affinity of the receptor-ligand interaction. Similarly, it isconceivable that the affinity of a mAb for a surface receptor coulddetermine the nature of intracellular signaling and whether the mAb maydeliver an agonist or an antagonist signal. The affinity-based nature ofmAb-mediated signaling may have an impact of its side-effect profile.Therefore, the desired affinity and desired functions of therapeuticmonoclonal antibodies need to be determined carefully by in vitro and invivo experimentation.

The desired Kd of a binding protein (e.g., an antibody) may bedetermined experimentally depending on the desired therapeutic outcome.In an embodiment parent antibodies with affinity (Kd) for a particularantigen equal to, or better than, the desired affinity of the DVD-Ig forthe same antigen are selected. The antigen binding affinity and kineticsare assessed by Biacore or another similar technique. In one embodiment,each parent antibody has a dissociation constant (Kd) to its antigenselected from the group consisting of: at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at mostabout 10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³M. First parentantibody from which VD1 is obtained and second parent antibody fromwhich VD2 is obtained may have similar or different affinity (K_(D)) forthe respective antigen. Each parent antibody has an on rate constant(Kon) to the antigen selected from the group consisting of: at leastabout 10²M⁻¹ s⁻¹; at least about 10³M⁻¹ s⁻¹; at least about 10⁴M⁻¹ s⁻¹;at least about 10⁵M⁻¹ s⁻¹; and at least about 10⁶M⁻¹ s⁻¹, as measured bysurface plasmon resonance. The first parent antibody from which VD1 isobtained and the second parent antibody from which VD2 is obtained mayhave similar or different on rate constant (Kon) for the respectiveantigen. In one embodiment, each parent antibody has an off rateconstant (Koff) to the antigen selected from the group consisting of: atmost about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; andat most about 10⁻⁶ s⁻¹, as measured by surface plasmon resonance. Thefirst parent antibody from which VD1 is obtained and the second parentantibody from which VD2 is obtained may have similar or different offrate constants (Koff) for the respective antigen.

B2. Potency

The desired affinity/potency of parental monoclonal antibodies willdepend on the desired therapeutic outcome. For example, forreceptor-ligand (R-L) interactions the affinity (kd) is equal to orbetter than the R-L kd (pM range). For simple clearance of a pathologiccirculating protein, the kd could be in low nM range e.g., clearance ofvarious species of circulating A-β peptide. In addition, the kd willalso depend on whether the target expresses multiple copies of the sameepitope e.g a mAb targeting conformational epitope in Aβ oligomers.

Where VDI and VD2 bind the same antigen, but distint epitopes, theDVD-Ig will contain 4 binding sites for the same antigen, thusincreasing avidity and thereby the apparent kd of the DVD-Ig. In anembodiment, parent antibodies with equal or lower kd than that desiredin the DVD-Ig are chosen. The affinity considerations of a parental mAbmay also depend upon whether the DVD-Ig contains four or more identicalantigen binding sites (i.e; a DVD-Ig from a single mAb). In this case,the apparent kd would be greater than the mAb due to avidity. SuchDVD-Igs can be employed for cross-linking surface receptor, increaseneutralization potency, enhance clearance of pathological proteins etc.

In an embodiment parent antibodies with neutralization potency forspecific antigen equal to or better than the desired neutralizationpotential of the DVD-Ig for the same antigen are selected. Theneutralization potency can be assessed by a target-dependent bioassaywhere cells of appropriate type produce a measurable signal (i.e.proliferation or cytokine production) in response to target stimulation,and target neutralization by the mAb can reduce the signal in adose-dependent manner.

B3. Biological Functions

Monoclonal antibodies can perform potentially several functions. Some ofthese functions are listed in Table 1. These functions can be assessedby both in vitro assays (e.g., cell-based and biochemical assays) and invivo animal models.

TABLE 1 Some Potential Applications For Therapeutic Antibodies Target(Class) Mechanism of Action (target) Soluble Neutralization of activity(e.g., a cytokine) (cytokines, other) Enhance clearance (e.g., Aβoligomers) Increase half-life (e.g., GLP 1) Cell Surface Agonist (e.g.,GLP1 R; EPO R; etc.) (Receptors, other) Antagonist (e.g., integrins;etc.) Cytotoxic (CD 20; etc.) Protein deposits Enhanceclearance/degradation (e.g., Aβ plaques, amyloid deposits)

MAbs with distinct functions described in the examples herein in Table 1can be selected to achieve desired therapeutic outcomes. Two or moreselected parent monoclonal antibodies can then be used in DVD-Ig formatto achieve two distinct functions in a single DVD-Ig molecule. Forexample, a DVD-Ig can be generated by selecting a parent mAb thatneutralizes function of a specific cytokine, and selecting a parent mAbthat enhances clearance of a pathological protein. Similarly, we canselect two parent monoclonal antibodies that recognize two differentcell surface receptors, one mAb with an agonist function on one receptorand the other mAb with an antagonist function on a different receptor.These two selected monoclonal antibodies each with a distinct functioncan be used to construct a single DVD-Ig molecule that will possess thetwo distinct functions (agonist and antagonist) of the selectedmonoclonal antibodies in a single molecule. Similarly, two antagonisticmonoclonal antibodies to cell surface receptors each blocking binding ofrespective receptor ligands (e.g., EGF and IGF) can be used in a DVD-Igformat. Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR)and a neutralizing anti-soluble mediator (e.g., anti-IGF1/2) mAb can beselected to make a DVD-Ig.

B4. Epitope Recognition:

Different regions of proteins may perform different functions. Forexample specific regions of a cytokine interact with the cytokinereceptor to bring about receptor activation whereas other regions of theprotein may be required for stabilizing the cytokine. In this instanceone may select a mAb that binds specifically to the receptor interactingregion(s) on the cytokine and thereby block cytokine-receptorinteraction. In some cases, for example certain chemokine receptors thatbind multiple ligands, a mAb that binds to the epitope (region onchemokine receptor) that interacts with only one ligand can be selected.In other instances, monoclonal antibodies can bind to epitopes on atarget that are not directly responsible for physiological functions ofthe protein, but binding of a mAb to these regions could eitherinterfere with physiological functions (steric hindrance) or alter theconformation of the protein such that the protein cannot function (mAbto receptors with multiple ligand which alter the receptor conformationsuch that none of the ligand can bind). Anti-cytokine monoclonalantibodies that do not block binding of the cytokine to its receptor,but block signal transduction have also been identified (e.g., 125-2H,an anti-IL-18 mAb).

Examples of epitopes and mAb functions include, but are not limited to,blocking Receptor-Ligand (R-L) interaction (neutralizing mAb that bindsR-interacting site); steric hindrance resulting in diminished or noR-binding. An Ab can bind the target at a site other than a receptorbinding site, but still interferes with receptor binding and functionsof the target by inducing conformational change and eliminate function(e.g., Xolair), binding to R but block signaling (125-2H).

In an embodiment, the parental mAb needs to target the appropriateepitope for maximum efficacy. Such epitope should be conserved in theDVD-Ig. The binding epitope of a mAb can be determined by severalapproaches, including co-crystallography, limited proteolysis ofmAb-antigen complex plus mass spectrometric peptide mapping (Legros V.et al 2000 Protein Sci. 9:1002-10), phage displayed peptide libraries(O'Connor K H et al 2005 J Immunol Methods. 299:21-35), as well asmutagenesis (Wu C. et al. 2003 J Immunol 170:5571-7).

B5. Physicochemical and Pharmaceutical Properties:

Therapeutic treatment with antibodies often requires administration ofhigh doses, often several mg/kg (due to a low potency on a mass basis asa consequence of a typically large molecular weight). In order toaccommodate patient compliance and to adequately address chronic diseasetherapies and outpatient treatment, subcutaneous (s.c.) or intramuscular(i.m.) administration of therapeutic mAbs is desirable. For example, themaximum desirable volume for s.c. administration is ˜1.0 mL, andtherefore, concentrations of >100 mg/mL are desirable to limit thenumber of injections per dose. In an embodiment, the therapeuticantibody is administered in one dose. The development of suchformulations is constrained, however, by protein-protein interactions(e.g., aggregation, which potentially increases immunogenicity risks)and by limitations during processing and delivery (e.g., viscosity).Consequently, the large quantities required for clinical efficacy andthe associated development constraints limit full exploitation of thepotential of antibody formulation and s.c. administration in high-doseregimens. It is apparent that the physicochemical and pharmaceuticalproperties of a protein molecule and the protein solution are of utmostimportance, e.g., stability, solubility and viscosity features.

B5.1. Stability:

A “stable” antibody formulation is one in which the antibody thereinessentially retains its physical stability and/or chemical stabilityand/or biological activity upon storage. Stability can be measured at aselected temperature for a selected time period. In an embodiment, theantibody in the formulation is stable at room temperature (about 30° C.)or at 40° C. for at least 1 month and/or stable at about 2-8° C. for atleast 1 year for at least 2 years. Furthermore, in an embodiment, theformulation is stable following freezing (to, e.g., −70° C.) and thawingof the formulation, hereinafter referred to as a “freeze/thaw cycle.” Inanother example, a “stable” formulation may be one wherein less thanabout 10% and less than about 5% of the protein is present as anaggregate in the formulation.

A DVD-Ig stable in vitro at various temperatures for an extended timeperiod is desirable. One can achieve this by rapid screening of parentalmAbs stable in vitro at elevated temperature, e.g., at 40° C. for 2-4weeks, and then assess stability. During storage at 2-8° C., the proteinreveals stability for at least 12 months, e.g., at least 24 months.Stability (% of monomeric, intact molecule) can be assessed usingvarious techniques such as cation exchange chromatography, sizeexclusion chromatography, SDS-PAGE, as well as bioactivity testing. Fora more comprehensive list of analytical techniques that may be employedto analyze covalent and conformational modifications see Jones, A. J. S.(1993) Analytical methods for the assessment of protein formulations anddelivery systems. In: Cleland, J. L.; Langer, R., editors. Formulationand delivery of peptides and proteins, 1^(st) edition, Washington, ACS,pg. 22-45; and Pearlman, R.; Nguyen, T. H. (1990) Analysis of proteindrugs. In: Lee, V. H., editor. Peptide and protein drug delivery, 1stedition, New York, Marcel Dekker, Inc., pg. 247-301.

Heterogeneity and aggregate formation: stability of the antibody may besuch that the formulation may reveal less than about 10%, and, in anembodiment, less than about 5%, in another embodiment, less than about2%, or, in an embodiment, within the range of 0.5% to 1.5% or less inthe GMP antibody material that is present as aggregate. Size exclusionchromatography is a method that is sensitive, reproducible, and veryrobust in the detection of protein aggregates.

In addition to low aggregate levels, the antibody must, in anembodiment, be chemically stable. Chemical stability may be determinedby ion exchange chromatography (e.g., cation or anion exchangechromatography), hydrophobic interaction chromatography, or othermethods such as isoelectric focusing or capillary electrophoresis. Forinstance, chemical stability of the antibody may be such that afterstorage of at least 12 months at 2-8° C. the peak representingunmodified antibody in a cation exchange chromatography may increase notmore than 20%, in an embodiment, not more than 10%, or, in anotherembodiment, not more than 5% as compared to the antibody solution priorto storage testing.

In an embodiment, the parent antibodies display structural integrity;correct disulfide bond formation, and correct folding: Chemicalinstability due to changes in secondary or tertiary structure of anantibody may impact antibody activity. For instance, stability asindicated by activity of the antibody may be such that after storage ofat least 12 months at 2-8° C. the activity of the antibody may decreasenot more than 50%, in an embodiment not more than 30%, or even not morethan 10%, or in an embodiment not more than 5% or 1% as compared to theantibody solution prior to storage testing. Suitable antigen-bindingassays can be employed to determine antibody activity.

B5.2. Solubility:

The “solubility” of a mAb correlates with the production of correctlyfolded, monomeric IgG. The solubility of the IgG may therefore beassessed by HPLC. For example, soluble (monomeric) IgG will give rise toa single peak on the HPLC chromatograph, whereas insoluble (e.g.,multimeric and aggregated) will give rise to a plurality of peaks. Aperson skilled in the art will therefore be able to detect an increaseor decrease in solubility of an IgG using routine HPLC techniques. For amore comprehensive list of analytical techniques that may be employed toanalyze solubility (see Jones, A. G. Dep. Chem. Biochem. Eng., Univ.Coll. London, London, UK. Editor(s): Shamlou, P. Ayazi. Process.Solid-Liq. Suspensions (1993), 93-117. Publisher: Butterworth-Heinemann,Oxford, UK and Pearlman, Rodney; Nguyen, Tue H, Advances in ParenteralSciences (1990), 4 (Pept. Protein Drug Delivery), 247-301). Solubilityof a therapeutic mAb is critical for formulating to high concentrationoften required for adequate dosing. As outlined herein, solubilitiesof >100 mg/mL may be required to accommodate efficient antibody dosing.For instance, antibody solubility may be not less than about 5 mg/mL inearly research phase, in an embodiment not less than about 25 mg/mL inadvanced process science stages, or in an embodiment not less than about100 mg/mL, or in an embodiment not less than about 150 mg/mL. It isobvious to a person skilled in the art that the intrinsic properties ofa protein molecule are important the physico-chemical properties of theprotein solution, e.g., stability, solubility, viscosity. However, aperson skilled in the art will appreciate that a broad variety ofexcipients exist that may be used as additives to beneficially impactthe characteristics of the final protein formulation. These excipientsmay include: (i) liquid solvents, cosolvents (e.g., alcohols such asethanol); (ii) buffering agents (e.g., phosphate, acetate, citrate,amino acid buffers); (iii) sugars or sugar alcohols (e.g., sucrose,trehalose, fructose, raffinose, mannitol, sorbitol, dextrans); (iv)surfactants (e.g., polysorbate 20, 40, 60, 80, poloxamers); (v)isotonicity modifiers (e.g., salts such as NaCl, sugars, sugaralcohols); and (vi) others (e.g., preservatives, chelating agents,antioxidants, chelating substances (e.g., EDTA), biodegradable polymers,carrier molecules (e.g., HSA, PEGs)

Viscosity is a parameter of high importance with regard to antibodymanufacture and antibody processing (e.g.,diafiltration/ultrafiltration), fill-finish processes (pumping aspects,filtration aspects) and delivery aspects (syringeability, sophisticateddevice delivery). Low viscosities enable the liquid solution of theantibody having a higher concentration. This enables the same dose maybe administered in smaller volumes. Small injection volumes inhere theadvantage of lower pain on injection sensations, and the solutions notnecessarily have to be isotonic to reduce pain on injection in thepatient. The viscosity of the antibody solution may be such that atshear rates of 100 (1/s) antibody solution viscosity is below 200 mPa s,in an embodiment below 125 mPa s, in another embodiment below 70 mPa s,and in yet another embodiment below 25 mPa s or even below 10 mPa s.

B 5.3. Production Efficiency

The generation of a DVD-Ig that is efficiently expressed in mammaliancells, such as Chinese hamster ovary cells (CHO), will in an embodimentrequire two parental monoclonal antibodies which are themselvesexpressed efficiently in mammalian cells. The production yield from astable mammalian line (i.e., CHO) should be above about 0.5 g/L, in anembodiment above about 1 g/L, and in another embodiment in the range ofabout 2-5 g/L or more (Kipriyanov S M, Little M. 1999 Mol Biotechnol.12:173-201; Carroll S, Al-Rubeai M. 2004 Expert Opin Biol Ther.4:1821-9).

Production of antibodies and Ig fusion proteins in mammalian cells isinfluenced by several factors. Engineering of the expression vector viaincorporation of strong promoters, enhancers and selection markers canmaximize transcription of the gene of interest from an integrated vectorcopy. The identification of vector integration sites that are permissivefor high levels of gene transcription can augment protein expressionfrom a vector (Wurm et al, 2004, Nature Biotechnology, 2004, Vol/Iss/Pg.22/11 (1393-1398)). Furthermore, levels of production are affected bythe ratio of antibody heavy and light chains and various steps in theprocess of protein assembly and secretion (Jiang et al. 2006,Biotechnology Progress, January-February 2006, vol. 22, no. 1, p.313-8).

B 6. Immunogenicity

Administration of a therapeutic mAb may results in certain incidence ofan immune response (ie, the formation of endogenous antibodies directedagainst the therapeutic mAb). Potential elements that might induceimmunogenicity should be analyzed during selection of the parentalmonoclonal antibodies, and steps to reduce such risk can be taken tooptimize the parental monoclonal antibodies prior to DVD-Igconstruction. Mouse-derived antibodies have been found to be highlyimmunogenic in patients. The generation of chimeric antibodies comprisedof mouse variable and human constant regions presents a logical nextstep to reduce the immunogenicity of therapeutic antibodies (Morrisonand Schlom, 1990). Alternatively, immunogenicity can be reduced bytransferring murine CDR sequences into a human antibody framework(reshaping/CDR grafting/humanization), as described for a therapeuticantibody by Riechmann et al., 1988. Another method is referred to as“resurfacing” or “veneering”, starting with the rodent variable lightand heavy domains, only surface-accessible framework amino acids arealtered to human ones, while the CDR and buried amino acids remain fromthe parental rodent antibody (Roguska et al., 1996). In another type ofhumanization, instead of grafting the entire CDRs, one technique graftsonly the “specificity-determining regions” (SDRs), defined as the subsetof CDR residues that are involved in binding of the antibody to itstarget (Kashmiri et al., 2005). This necessitates identification of theSDRs either through analysis of available three-dimensional structuresof antibody-target complexes or mutational analysis of the antibody CDRresidues to determine which interact with the target. Alternatively,fully human antibodies may have reduced immunogenicity compared tomurine, chimeric or humanized antibodies.

Another approach to reduce the immunogenicity of therapeutic antibodiesis the elimination of certain specific sequences that are predicted tobe immunogenic. In one approach, after a first generation biologic hasbeen tested in humans and found to be unacceptably immunogenic, theB-cell epitopes can be mapped and then altered to avoid immunedetection. Another approach uses methods to predict and remove potentialT-cell epitopes. Computational methods have been developed to scan andto identify the peptide sequences of biologic therapeutics with thepotential to bind to MHC proteins (Desmet et al., 2005). Alternatively ahuman dendritic cell-based method can be used to identify CD4⁺ T-cellepitopes in potential protein allergens (Stickler et al., 2005; S. L.Morrison and J. Schlom, Important Adv. Oncol. (1990), pp. 3-18;Riechmann, L., Clark, M., Waldmann, H. and Winter, G. “Reshaping humanantibodies for therapy.” Nature (1988) 332: 323-327; Roguska-M-A,Pedersen-J-T, Henry-A-H, Searle-S-M, Roja-C-M, Avery-B, Hoffee-M,Cook-S, Lambert-J-M, Blättler-W-A, Rees-A-R, Guild-B-C. A comparison oftwo murine mAbs humanized by CDR-grafting and variable domainresurfacing. Protein engineering, {Protein-Eng}, 1996, vol. 9, p.895-904; Kashmiri-Syed-V-S, De-Pascalis-Roberto, Gonzales-Noreen-R,Schlom-Jeffrey. SDR grafting—a new approach to antibody humanization.Methods (San Diego Calif.), {Methods}, May 2005, vol. 36, no. 1, p.25-34; Desmet-Johan, Meersseman-Geert, Boutonnet-Nathalie,Pletinckx-Jurgen, De-Clercq-Krista, Debulpaep-Maja, Braeckman-Tessa,Lasters-Ignace. Anchor profiles of HLA-specific peptides: analysis by anovel affinity scoring method and experimental validation. Proteins,2005, vol. 58, p. 53-69; Stickler-M-M, Estell-D-A, Harding-F-A. CD4+T-cell epitope determination using unexposed human donor peripheralblood mononuclear cells. Journal of immunotherapy 2000, vol. 23, p.654-60.)

B 7. In Vivo Efficacy

To generate a DVD-Ig molecule with desired in vivo efficacy, it isimportant to generate and select mAbs with similarly desired in vivoefficacy when given in combination. However, in some instances theDVD-Ig may exhibit in vivo efficacy that cannot be achieved with thecombination of two separate mAbs. For instance, a DVD-Ig may bring twotargets in close proximity leading to an activity that cannot beachieved with the combination of two separate mAbs. Additional desirablebiological functions are described herein in section B 3. Parentantibodies with characteristics desirable in the DVD-Ig molecule may beselected based on factors such as pharmacokinetic t½; tissuedistribution; soluble versus cell surface targets; and targetconcentration—soluble/density—surface.

B 8. In Vivo Tissue Distribution

To generate a DVD-Ig molecule with desired in vivo tissue distribution,in an embodiment parent mAbs with similar desired in vivo tissuedistribution profile must be selected. Alternatively, based on themechanism of the dual-specific targeting strategy, it may at other timesnot be required to select parent mAbs with the similarly desired in vivotissue distribution when given in combination. For instance, in the caseof a DVD-Ig in which one binding component targets the DVD-Ig to aspecific site thereby bringing the second binding component to the sametarget site. For example, one binding specificity of a DVD-Ig couldtarget pancreas (islet cells) and the other specificity could bring GLP1to the pancreas to induce insulin.

B 9. Isotype:

To generate a DVD-Ig molecule with desired properties including, but notlimited to, Isotype, Effector functions and the circulating half-life,in an embodiment parent mAbs with appropriate Fc-effector functionsdepending on the therapeutic utility and the desired therapeuticend-point are selected. There are five main heavy-chain classes orisotypes some of which have several sub-types and these determine theeffector functions of an antibody molecule. These effector functionsreside in the hinge region, CH2 and CH3 domains of the antibodymolecule. However, residues in other parts of an antibody molecule mayhave effects on effector functions as well. The hinge region Fc-effectorfunctions include: (i) antibody-dependent cellular cytotoxicity, (ii)complement (C1q) binding, activation and complement-dependentcytotoxicity (CDC), (iii) phagocytosis/clearance of antigen-antibodycomplexes, and (iv) cytokine release in some instances. TheseFc-effector functions of an antibody molecule are mediated through theinteraction of the Fc-region with a set of class-specific cell surfacereceptors. Antibodies of the IgG1 isotype are most active while IgG2 andIgG4 having minimal or no effector functions. The effector functions ofthe IgG antibodies are mediated through interactions with threestructurally homologous cellular Fc receptor types (and sub-types)(FcgR1, FcgRII and FcgRIII). These effector functions of an IgG1 can beeliminated by mutating specific amino acid residues in the lower hingeregion (e.g., L234A, L235A) that are required for FcgR and C1q binding.Amino acid residues in the Fc region, in particular the CH2-CH3 domains,also determine the circulating half-life of the antibody molecule. ThisFc function is mediated through the binding of the Fc-region to theneonatal Fc receptor (FcRn) which is responsible for recycling ofantibody molecules from the acidic lysosomes back to the generalcirculation.

Whether a mAb should have an active or an inactive isotype will dependon the desired therapeutic end-point for an antibody. Some examples ofusage of isotypes and desired therapeutic outcome are listed below:

-   -   a) If the desired end-point is functional neutralization of a        soluble cytokine then an inactive isotype may be used;    -   b) If the desired out-come is clearance of a pathological        protein an active isotype may be used;    -   c) If the desired out-come is clearance of protein aggregates an        active isotype may be used;    -   d) If the desired outcome is to antagonize a surface receptor an        inactive isotype is used (Tysabri, IgG4; OKT3, mutated IgG1);    -   e) If the desired outcome is to eliminate target cells an active        isotype is used (Herceptin, IgG1 (and with enhanced effector        functions); and    -   f) If the desired outcome is to clear proteins from circulation        without entering the CNS an IgM isotype may be used (e.g.,        clearing circulating Ab peptide species).

The Fc effector functions of a parental mAb can be determined by variousin vitro methods well known in the art.

As discussed, the selection of isotype, and thereby the effectorfunctions will depend upon the desired therapeutic end-point. In caseswhere simple neutralization of a circulating target is desired, forexample blocking receptor-ligand interactions, the effector functionsmay not be required. In such instances isotypes or mutations in theFc-region of an antibody that eliminate effector functions aredesirable. In other instances where elimination of target cells is thetherapeutic end-point, for example elimination of tumor cells, isotypesor mutations or de-fucosylation in the Fc-region that enhance effectorfunctions are desirable (Presta G L, Adv. Drug Delivery Rev. 58:640-656,2006; Satoh M., Iida S., Shitara K. Expert Opinion Biol. Ther.6:1161-1173, 2006). Similarly, depending up on the therapeutic utility,the circulating half-life of an antibody molecule can bereduced/prolonged by modulating antibody-FcRn interactions byintroducing specific mutations in the Fc region (Dall'Acqua W F, KienerP A, Wu H. J. Biol. Chem. 281:23514-23524, 2006; Petkova S B., AkileshS., Sproule T J. et al. Internat. Immunol. 18:1759-1769, 2006; VaccaroC., Bawdon R., Wanjie S et al. PNAS 103:18709-18714, 2007).

The published information on the various residues that influence thedifferent effector functions of a normal therapeutic mAb may need to beconfirmed for DVD-Ig. It may be possible that in a DVD-Ig formatadditional (different) Fc-region residues, other than those identifiedfor the modulation of monoclonal antibody effector functions, may beimportant.

Overall, the decision as to which Fc-effector functions (isotype) willbe critical in the final DVD-Ig format will depend up on the diseaseindication, therapeutic target, desired therapeutic end-point and safetyconsiderations. Listed below are exemplary appropriate heavy chain andlight chain constant regions including, but not limited to:

-   -   IgG1—allotype: Glmz    -   IgG1 mutant—A234, A235    -   IgG2—allotype: G2m(n-)    -   Kappa—Km3    -   Lambda

Fc Receptor and C1q Studies: The possibility of unwantedantibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) by antibody complexing to anyoverexpressed target on cell membranes can be abrogated by the (forexample, L234A, L235A) hinge-region mutations. These substituted aminoacids, present in the IgG1 hinge region of mAb, are expected to resultin diminished binding of mAb to human Fc receptors (but not FcRn), asFcgR binding is thought to occur within overlapping sites on the IgG1hinge region. This feature of mAb may lead to an improved safety profileover antibodies containing a wild-type IgG. Binding of mAb to human Fcreceptors can be determined by flow cytometry experiments using celllines (e.g., THP-1, K562) and an engineered CHO cell line that expressesFcgRIIb (or other FcgRs). Compared to IgG1 control monoclonalantibodies, mAb show reduced binding to FcgRI and FcgRIIa whereasbinding to FcgRIIb is unaffected. The binding and activation of C1q byantigen/IgG immune complexes triggers the classical complement cascadewith consequent inflammatory and/or immunoregulatory responses. The C1qbinding site on IgGs has been localized to residues within the IgG hingeregion. C1q binding to increasing concentrations of mAb was assessed byC1q ELISA. The results demonstrate that mAb is unable to bind to C1q, asexpected when compared to the binding of a wildtype control IgG1.Overall, the L234A, L235A hinge region mutation abolishes binding of mAbto FcgRI, FcgRIIa and C1q but does not impact the interaction of mAbwith FcgRIIb. This data suggests that in vivo, mAb with mutant Fc willinteract normally with the inhibitory FcgRIIb but will likely fail tointeract with the activating FcgRI and FcgRIIa receptors or C1q.

Human FcRn Binding:

The neonatal receptor (FcRn) is responsible for transport of IgG acrossthe placenta and to control the catabolic half-life of the IgGmolecules. It might be desirable to increase the terminal half-life ofan antibody to improve efficacy, to reduce the dose or frequency ofadministration, or to improve localization to the target. Alternatively,it might be advantageous to do the converse that is, to decrease theterminal half-life of an antibody to reduce whole body exposure or toimprove the target-to-non-target binding ratios. Tailoring theinteraction between IgG and its salvage receptor, FcRn, offers a way toincrease or decrease the terminal half-life of IgG. Proteins in thecirculation, including IgG, are taken up in the fluid phase throughmicropinocytosis by certain cells, such as those of the vascularendothelia. IgG can bind FcRn in endosomes under slightly acidicconditions (pH 6.0-6.5) and can recycle to the cell surface, where it isreleased under almost neutral conditions (pH 7.0-7.4). Mapping of theFc-region-binding site on FcRn80, 16, 17 showed that two histidineresidues that are conserved across species, His310 and His435, areresponsible for the pH dependence of this interaction. Usingphage-display technology, a mouse Fc-region mutation that increasesbinding to FcRn and extends the half-life of mouse IgG was identified(see Victor, G. et al.; Nature Biotechnology (1997), 15(7), 637-640).Fc-region mutations that increase the binding affinity of human IgG forFcRn at pH 6.0, but not at pH 7.4, have also been identified (seeDall'Acqua William F, et al., Journal of Immunology (2002), 169(9),5171-80). Moreover, in one case, a similar pH-dependent increase inbinding (up to 27-fold) was also observed for rhesus FcRn, and thisresulted in a twofold increase in serum half-life in rhesus monkeyscompared with the parent IgG (see Hinton, Paul R. et al., Journal ofBiological Chemistry (2004), 279(8), 6213-6216). These findings indicatethat it is feasible to extend the plasma half-life of antibodytherapeutics by tailoring the interaction of the Fc region with FcRn.Conversely, Fc-region mutations that attenuate interaction with FcRn canreduce antibody half-life.

B.10 Pharmacokinetics (PK):

To generate a DVD-Ig molecule with desired pharmacokinetic profile, inan embodiment parent mAbs with the similarly desired pharmacokineticprofile are selected. One consideration is that immunogenic response tomonoclonal antibodies (ie, HAHA, human anti-human antibody response;HACA, human anti-chimeric antibody response) further complicates thepharmacokinetics of these therapeutic agents. In an embodiment,monoclonal antibodies with minimal or no immunogenicity are used forconstructing DVD-Ig molecules such that the resulting DVD-Igs will alsohave minimal or no immunogenicity. Some of the factors that determinethe PK of a mAb include, but are not limited to, Intrinsic properties ofthe mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fcfunctions.

The PK profile of selected parental monoclonal antibodies can be easilydetermined in rodents as the PK profile in rodents correlates well with(or closely predicts) the PK profile of monoclonal antibodies incynomolgus monkey and humans. The PK profile is determined as describedin Example section 1.2.2.3.A.

After the parental monoclonal antibodies with desired PK characteristics(and other desired functional properties as discussed herein) areselected, the DVD-Ig is constructed. As the DVD-Ig molecules contain twoantigen-binding domains from two parental monoclonal antibodies, the PKproperties of the DVD-Ig are assessed as well. Therefore, whiledetermining the PK properties of the DVD-Ig, PK assays may be employedthat determine the PK profile based on functionality of bothantigen-binding domains derived from the 2 parent monoclonal antibodies.The PK profile of a DVD-Ig can be determined as described in Example1.2.2.3.A. Additional factors that may impact the PK profile of DVD-Iginclude the antigen-binding domain (CDR) orientation; Linker size; andFc/FcRn interactions. PK characteristics of parent antibodies can beevaluated by assessing the following parameters: absorption,distribution, metabolism and excretion.

Absorption: To date, administration of therapeutic monoclonal antibodiesis via parenteral routes (e.g., intravenous [IV], subcutaneous [SC], orintramuscular [IM]). Absorption of a mAb into the systemic circulationfollowing either SC or IM administration from the interstitial space isprimarily through the lymphatic pathway. Saturable, presystemic,proteolytic degradation may result in variable absolute bioavailabilityfollowing extravascular administration. Usually, increases in absolutebioavailability with increasing doses of monoclonal antibodies may beobserved due to saturated proteolytic capacity at higher doses. Theabsorption process for a mAb is usually quite slow as the lymph fluiddrains slowly into the vascular system, and the duration of absorptionmay occur over hours to several days. The absolute bioavailability ofmonoclonal antibodies following SC administration generally ranges from50% to 100%. In the case of a transport-mediating structure at theblood-brain barrier targeted by the DVD-Ig construct, circulation timesin plasma may be reduced due to enhanced trans-cellular transport at theblood brain barrier (BBB) into the CNS compartment, where the DVD-Ig isliberated to enable interaction via its second antigen recognition site.

Distribution:

Following IV administration, monoclonal antibodies usually follow abiphasic serum (or plasma) concentration-time profile, beginning with arapid distribution phase, followed by a slow elimination phase. Ingeneral, a biexponential pharmacokinetic model best describes this kindof pharmacokinetic profile. The volume of distribution in the centralcompartment (Vc) for a mAb is usually equal to or slightly larger thanthe plasma volume (2-3 liters). A distinct biphasic pattern in serum(plasma) concentration versus time profile may not be apparent withother parenteral routes of administration, such as IM or SC, because thedistribution phase of the serum (plasma) concentration-time curve ismasked by the long absorption portion. Many factors, includingphysicochemical properties, site-specific and target-oriented receptormediated uptake, binding capacity of tissue, and mAb dose can influencebiodistribution of a mAb. Some of these factors can contribute tononlinearity in biodistribution for a mAb.

Metabolism and Excretion:

Due to the molecular size, intact monoclonal antibodies are not excretedinto the urine via kidney. They are primarily inactivated by metabolism(e.g., catabolism). For IgG-based therapeutic monoclonal antibodies,half-lives typically ranges from hours or 1-2 days to over 20 days. Theelimination of a mAb can be affected by many factors, including, but notlimited to, affinity for the FcRn receptor, immunogenicity of the mAb,the degree of glycosylation of the mAb, the susceptibility for the mAbto proteolysis, and receptor-mediated elimination.

B.11 Tissue Cross-Reactivity Pattern on Human and Tox Species:

Identical staining pattern suggests that potential human toxicity can beevaluated in tox species. Tox species are those animal in whichunrelated toxicity is studied.

The individual antibodies are selected to meet two criteria. (1) Tissuestaining appropriate for the known expression of the antibody target.(2) Similar staining pattern between human and tox species tissues fromthe same organ.

Criterion 1: Immunizations and/or antibody selections typically employrecombinant or synthesized antigens (proteins, carbohydrates or othermolecules). Binding to the natural counterpart and counterscreen againstunrelated antigens are often part of the screening funnel fortherapeutic antibodies. However, screening against a multitude ofantigens is often unpractical. Therefore tissue cross-reactivity studieswith human tissues from all major organs serve to rule out unwantedbinding of the antibody to any unrelated antigens.

Criterion 2: Comparative tissue cross reactivity studies with human andtox species tissues (cynomolgus monkey, dog, possibly rodents andothers, the same 36 or 37 tissues are being tested as in the humanstudy) help to validate the selection of a tox species. In the typicaltissue cross-reactivity studies on frozen tissues sections therapeuticantibodies may demonstrate the expected binding to the known antigenand/or to a lesser degree binding to tissues based either on low levelinteractions (unspecific binding, low level binding to similar antigens,low level charge based interactions etc.). In any case the most relevanttoxicology animal species is the one with the highest degree ofcoincidence of binding to human and animal tissue.

Tissue cross reactivity studies follow the appropriate regulatoryguidelines including EC CPMP Guideline 111/5271/94 “Production andquality control of mAbs” and the 1997 US FDA/CBER “Points to Consider inthe Manufacture and Testing of Monoclonal Antibody Products for HumanUse”. Cryosections (5 μm) of human tissues obtained at autopsy or biopsywere fixed and dried on object glass. The peroxidase staining of tissuesections was performed, using the avidin-biotin system. FDA's Guidance“Points to Consider in the Manufacture and Testing of MonoclonalAntibody Products for Human Use”. Relevant references include Clarke J2004, Boon L. 2002a, Boon L 2002b, Ryan A 1999.

Tissue cross reactivity studies are often done in two stages, with thefirst stage including cryosections of 32 tissues (typically: AdrenalGland, Gastrointestinal Tract, Prostate, Bladder, Heart, SkeletalMuscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord,Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex,Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium, Parathyroid,Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from onehuman donor. In the second phase a full cross reactivity study isperformed with up to 38 tissues (including adrenal, blood, blood vessel,bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart,kidney, large intestine, liver, lung, lymph node, breast mammary gland,ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary,placenta, prostate, salivary gland, skin, small intestine, spinal cord,spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus) from 3 unrelated adults. Studiesare done typically at minimally two dose levels.

The therapeutic antibody (i.e. test article) and isotype matched controlantibody may be biotinylated for avidin-biotin complex (ABC) detection;other detection methods may include tertiary antibody detection for aFITC (or otherwise) labeled test article, or precomplexing with alabeled anti-human IgG for an unlabeled test article.

Briefly, cryosections (about 5 μm) of human tissues obtained at autopsyor biopsy are fixed and dried on object glass. The peroxidase stainingof tissue sections is performed, using the avidin-biotin system. First(in case of a precomplexing detection system), the test article isincubated with the secondary biotinylated anti-human IgG and developedinto immune complex. The immune complex at the final concentrations of 2and 10 μg/mL of test article is added onto tissue sections on objectglass and then the tissue sections were reacted for 30 minutes with aavidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine),a substrate for the peroxidase reaction, was applied for 4 minutes fortissue staining. Antigen-Sepharose beads are used as positive controltissue sections.

Any specific staining is judged to be either an expected (e.g.,consistent with antigen expression) or unexpected reactivity based uponknown expression of the target antigen in question. Any staining judgedspecific is scored for intensity and frequency. Antigen or serumcompetion or blocking studies can assist further in determining whetherobserved staining is specific or nonspecific.

If two selected antibodies are found to meet the selectioncriteria—appropriate tissue staining, matching staining between humanand toxicology animal specific tissue—they can be selected for DVD-Iggeneration.

The tissue cross reactivity study has to be repeated with the finalDVD-Ig construct, but while these studies follow the same protocol asoutline herein, they are more complex to evaluate because any bindingcan come from any of the two parent antibodies, and any unexplainedbinding needs to be confirmed with complex antigen competition studies.

It is readily apparent that the complex undertaking of tissuecrossreactivity studies with a multispecific molecule like a DVD-Ig isgreatly simplified if the two parental antibodies are selected for (1)lack of unexpected tissue cross reactivity findings and (2) forappropriate similarity of tissue cross reactivity findings between thecorresponding human and toxicology animal species tissues.

B.12 Specificity and Selectivity:

To generate a DVD-Ig molecule with desired specificity and selectivity,one needs to generate and select parent mAbs with the similarly desiredspecificity and selectivity profile.

Binding studies for specificity and selectivity with a DVD-Ig can becomplex due to the four or more binding sites, two each for eachantigen. Briefly, binding studies using ELISA, BIAcore KinExA or otherinteraction studies with a DVD-Ig need to monitor the binding of one,two or more antigens to the DVD-Ig molecule. While BIAcore technologycan resolve the sequential, independent binding of multiple antigens,more traditional methods including ELISA or more modern techniques likeKinExA cannot. Therefore careful characterization of each parentantibody is critical. After each individual antibody has beencharacterized for specificity, confirmation of specificity retention ofthe individual binding sites in the DVD-Ig molecule is greatlysimplified.

It is readily apparent that the complex undertaking of determining thespecificity of a DVD-Ig is greatly simplified if the two parentalantibodies are selected for specificity prior to being combined into aDVD-Ig.

Antigen—antibody interaction studies can take many forms, including manyclassical protein protein interaction studies, including ELISA (Enzymelinked immunosorbent assay), Mass spectrometry, chemical cross linking,SEC with light scattering, equilibrium dialysis, gel permeation,ultrafiltration, gel chromatography, large-zone analytical SEC,micropreparative ultracentrigugation (sedimentation equilibrium),spectroscopic methods, titration microcalorimetry, sedimentationequilibrium (in analytical ultracentrifuge), sedimentation velocity (inanalytical centrifuge), surface plasmon resonance (including BIAcore).Relevant references include “Current Protocols in Protein Science”, JohnE. Coligan, Ben M. Dunn, David W. Speicher, Paul T, Wingfield (eds.)Volume 3, chapters 19 and 20, published by John Wiley & Sons Inc., andreferences included therein and “Current Protocols in Immunology”, JohnE. Coligan, Barbara E. Bierer, David H. Margulies, Ethan M. Shevach,Warren Strober (eds.) published by John Wiley & Sons Inc and relevantreferences included therein.

Cytokine Release in Whole Blood: The interaction of mAb with human bloodcells can be investigated by a cytokine release assay (Wing, M. G.Therapeutic Immunology (1995), 2(4), 183-190; “Current Protocols inPharmacology”, S. J. Enna, Michael Williams, John W. Ferkany, TerryKenakin, Paul Moser, (eds.) published by John Wiley & Sons Inc;Madhusudan, S. Clinical Cancer Research (2004), 10(19), 6528-6534; Cox,J. Methods (2006), 38(4), 274-282; Choi, I. European Journal ofImmunology (2001), 31(1), 94-106). Briefly, various concentrations ofmAb are incubated with human whole blood for 24 hours. The concentrationtested should cover a wide range including final concentrationsmimicking typical blood levels in patients (including but not limited to100 ng/ml-100 μg/ml). Following the incubation, supernatants and celllysates were analyzed for the presence of IL-1Rα, TNF-α, IL-1b, IL-6 andIL-8. Cytokine concentration profiles generated for mAb were compared toprofiles produced by a negative human IgG control and a positive LPS orPHA control. The cytokine profile displayed by mAb from both cellsupernatants and cell lysates was comparable to control human IgG. In anembodiment, the monoclonal antibody does not interact with human bloodcells to spontaneously release inflammatory cytokines.

Cytokine release studies for a DVD-Ig are complex due to the four ormore binding sites, two each for each antigen. Briefly, cytokine releasestudies as described herein measure the effect of the whole DVD-Igmolecule on whole blood or other cell systems, but can resolve whichportion of the molecule causes cytokine release. Once cytokine releasehas been detected, the purity of the DVD-Ig preparation has to beascertained, because some co-purifying cellular components can causecytokine release on their own. If purity is not the issue, fragmentationof DVD-Ig (including but not limited to removal of Fc portion,separation of binding sites etc.), binding site mutagenesis or othermethods may need to be employed to deconvolute any observations. It isreadily apparent that this complex undertaking is greatly simplified ifthe two parental antibodies are selected for lack of cytokine releaseprior to being combined into a DVD-Ig.

B.13 Cross Reactivity to Other Species for Toxicological Studies:

In an embodiment, the individual antibodies selected with sufficientcross-reactivity to appropriate tox species, for example, cynomolgusmonkey. Parental antibodies need to bind to orthologous species target(i.e. cynomolgus monkey) and elicit appropriate response (modulation,neutralization, activation). In an embodiment, the cross-reactivity(affinity/potency) to orthologous species target should be within10-fold of the human target. In practice, the parental antibodies areevaluated for multiple species, including mouse, rat, dog, monkey (andother non-human primates), as well as disease model species (i.e. sheepfor asthma model). The acceptable cross-reactivity to tox species fromthe perantal monoclonal antibodies allows future toxicology studies ofDVD-Ig-Ig in the same species. For that reason, the two parentalmonoclonal antibodies should have acceptable cross-reactivity for acommon tox species therefore allowing toxicology studies of DVD-Ig inthe same species.

Parent mAbs may be selected from various mAbs capable of bindingspecific targets and well known in the art. These include, but are notlimited to anti-TNF antibody (U.S. Pat. No. 6,258,562), anti-IL-12and/or anti-IL-12p40 antibody (U.S. Pat. No. 6,914,128); anti-IL-18antibody (US 2005/0147610 A1), anti-05, anti-CBL, anti-CD147,anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L,anti CD-40 (e.g., see WO2007124299) anti-Id, anti-ICAM-1, anti-CXCL13,anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-HGF, anti-cMet, anti DLL-4,anti-NPR1, anti-PLGF, anti-ErbB3, anti-E-selectin, anti-Fact VII,anti-Her2/neu, anti-F gp, anti-CD11/18, anti-CD14, anti-ICAM-3,anti-RON, anti CD-19, anti-CD80 (e.g., see WO2003039486, anti-CD4,anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52,anti-HLA DR, anti-CD22 (e.g., see U.S. Pat. No. 5,789,554), anti-CD20,anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B,anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE,anti-CD25, anti-CD33, anti-HLA, anti-IGF1,2, anti IGFR,anti-VNRintegrin, anti-IL-1alpha, anti-IL-1 beta, anti-IL-1 receptor,anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5receptor, anti-IL-6, anti-IL-6R, RANKL, NGF, DKK, alphaVbeta3, IL-17A,anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, andanti-IL-23; IL-23p19; (see Presta L G. 2005 Selection, design, andengineering of therapeutic antibodies J Allergy Clin Immunol. 116:731-6and http://www.path.cam.ac.uk/˜mrc7/humanisation/antibodies.html).

Parent mAbs may also be selected from various therapeutic antibodiesapproved for use, in clinical trials, or in development for clinicaluse. Such therapeutic antibodies include, but are not limited to,rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat.No. 5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”), trastuzumab (Herceptin®,Genentech) (see for example U.S. Pat. No. 5,677,171), a humanizedanti-Her2/neu antibody approved to treat breast cancer; pertuzumab(rhuMab-2C4, Omnitarg®), currently being developed by Genentech; ananti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab(Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210), achimeric anti-EGFR antibody in clinical trials for a variety of cancers;ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed byAbgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No. 10/172,317), currentlybeing developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (MerckKGaA) (U.S. Pat. No. 5,558,864; Murthy et al. 1987, Arch BiochemBiophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem.35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83);ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi etal., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al.,1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br JCancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer,105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro deImmunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institue for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO0162931A2); and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab(Campath®, Millenium), a humanized mAb currently approved for treatmentof B-cell chronic lymphocytic leukemia; muromonab-CD3 (OrthocloneOKT3®), an anti-CD3 antibody developed by Ortho Biotech/Johnson &Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibodydeveloped by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), ananti-CD33 (p67 protein) antibody developed by Celltech/Wyeth, alefacept(Amevive®), an anti-LFA-3 Fc fusion developed by Biogen), abciximab(ReoPro®), developed by Centocor/Lilly, basiliximab (Simulect®),developed by Novartis, palivizumab (Synagis®), developed by Medimmune,infliximab (Remicade®), an anti-TNFalpha antibody developed by Centocor,adalimumab (Humira®), an anti-TNFalpha antibody developed by Abbott,Humicade®, an anti-TNFalpha antibody developed by Celltech, golimumab(CNTO-148), a fully human TNF antibody developed by Centocor, etanercept(Enbrel®), an p75 TNF receptor Fc fusion developed by Immunex/Amgen,lenercept, an p55TNF receptor Fc fusion previously developed by Roche,ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, ananti-IL8 antibody being developed by Abgenix, ABX-MA1, an anti-MUC18antibody being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), ananti-MUC1 in development by Antisoma, Therex (R1550), an anti-MUC1antibody being developed by Antisoma, AngioMab (AS1405), being developedby Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407)being developed by Antisoma, Antegren® (natalizumab), ananti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developedby Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed byBiogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibodybeing developed by Biogen, CAT-152, an anti-TGF-β2 antibody beingdeveloped by Cambridge Antibody Technology, ABT 874 (J695), ananti-IL-12 p40 antibody being developed by Abbott, CAT-192, ananti-TGFβ1 antibody being developed by Cambridge Antibody Technology andGenzyme, CAT-213, an anti-Eotaxinl antibody being developed by CambridgeAntibody Technology, LymphoStat-B® an anti-Blys antibody being developedby Cambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin®bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair® (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva® (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide® (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem® (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by Morph® Sys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF®, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-a 501 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) ahumanized anti-IgE antibody developed by Genentech and Novartis, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma, all ofthe herein-cited references in this paragraph are expressly incorporatedherein by reference. In another embodiment, the therapeutics includeKRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for CancerResearch); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha Vfβ3integrin, Medimmune); volociximab (alpha Vβ1 integrin, Biogen/PDL);Human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecificCD19×CD3, Medimmune); 4G7xH22 (Bispecific BcellxFcgammaRl, Medarex/MerckKGa); rM28 (Bispecific CD28×MAPG, US Patent No. EP1444268); MDX447 (EMD82633) (Bispecific CD64×EGFR, Medarex); Catumaxomab (removab)(Bispecific EpCAM×anti-CD3, Trion/Fres); Ertumaxomab (bispecificHER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx);Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2,Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist,Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507)(CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab(Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics);Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen);muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma);HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex);SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, SeattleGenentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40,Novartis); SGN-40 (CD40, Seattle Genentics); Campathlh (Alemtuzumab)(CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38,Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093/D93)(cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab(MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab,CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist,Human Genome Science/Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab(DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab)(DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone);IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab(Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab);CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201)(Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor);MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3.Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb,Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma);AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871(IGF-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen);Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor);Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193(Lewis (γ), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTBR,Biogen); HuHMFG1 (MUC1, Antisoma/NCI); RAV12 (N-linked carbohydrateepitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP),University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538)(PD1, Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa,Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA,Cornell Research Foundation); muJ591 (PSMA, Cornell ResearchFoundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab(Remicade) (TNFα, Centocor); A27.15 (transferrin receptor, SalkInstitute, INSERN WO 2005/111082); E2.3 (transferrin receptor, SalkInstitute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF,Tsukuba Research Lab-WO/2000/034337, University of Texas); IMC-18F1(VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).

B. Construction of DVD Molecules:

The dual variable domain immunoglobulin (DVD-Ig) molecule is designedsuch that two different light chain variable domains (VL) from the twodifferent parent monoclonal antibodies are linked in tandem directly orvia a short linker by recombinant DNA techniques, followed by the lightchain constant domain. Similarly, the heavy chain comprises twodifferent heavy chain variable domains (VH) linked in tandem, followedby the constant domain CH1 and Fc region (FIG. 1A).

The variable domains can be obtained using recombinant DNA techniquesfrom a parent antibody generated by any one of the methods describedherein. In an embodiment, the variable domain is a murine heavy or lightchain variable domain. In another embodiment, the variable domain is aCDR grafted or a humanized variable heavy or light chain domain. In anembodiment, the variable domain is a human heavy or light chain variabledomain.

In one embodiment the first and second variable domains are linkeddirectly to each other using recombinant DNA techniques. In anotherembodiment the variable domains are linked via a linker sequence. In anembodiment, two variable domains are linked. Three or more variabledomains may also be linked directly or via a linker sequence. Thevariable domains may bind the same antigen or may bind differentantigens. DVD molecules of the invention may include one immunoglobulinvariable domain and one non-immunoglobulin variable domain such asligand binding domain of a receptor, active domain of an enzyme. DVDmolecules may also comprise 2 or more non-Ig domains.

The linker sequence may be a single amino acid or a polypeptidesequence. In an embodiment, the linker sequences are selected from thegroup consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV(SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4);SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7);RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9);SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP(SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14);QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ IDNO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP(SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); and GHEAAAVMQVQYPAS (SEQ IDNO: 26). The choice of linker sequences is based on crystal structureanalysis of several Fab molecules. There is a natural flexible linkagebetween the variable domain and the CH1/CL constant domain in Fab orantibody molecular structure. This natural linkage comprisesapproximately 10-12 amino acid residues, contributed by 4-6 residuesfrom C-terminus of V domain and 4-6 residues from the N-terminus ofCL/CH1 domain. DVD Igs of the invention were generated using N-terminal5-6 amino acid residues, or 11-12 amino acid residues, of CL or CH1 aslinker in light chain and heavy chain of DVD-Ig, respectively. TheN-terminal residues of CL or CH1 domains, particularly the first 5-6amino acid residues, adopt a loop conformation without strong secondarystructures, therefore can act as flexible linkers between the twovariable domains. The N-terminal residues of CL or CH1 domains arenatural extension of the variable domains, as they are part of the Igsequences, therefore minimize to a large extent any immunogenicitypotentially arising from the linkers and junctions.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains; the light chain linkers canbe from Cκ or Cλ; and the heavy chain linkers can be derived from CH1 ofany isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ.Linker sequences may also be derived from other proteins such as Ig-likeproteins, (e.g.TCR, FcR, KIR); G/S based sequences (e.g G4S repeats; SEQID NO: 27); hinge region-derived sequences; and other natural sequencesfrom other proteins.

In an embodiment a constant domain is linked to the two linked variabledomains using recombinant DNA techniques. In an embodiment, sequencecomprising linked heavy chain variable domains is linked to a heavychain constant domain and sequence comprising linked light chainvariable domains is linked to a light chain constant domain. In anembodiment, the constant domains are human heavy chain constant domainand human light chain constant domain respectively. In an embodiment,the DVD heavy chain is further linked to an Fc region. The Fc region maybe a native sequence Fc region, or a variant Fc region. In anotherembodiment, the Fc region is a human Fc region. In another embodimentthe Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM,IgE, or IgD.

In another embodiment two heavy chain DVD polypeptides and two lightchain DVD polypeptides are combined to form a DVD-Ig molecule. Table 2lists amino acid sequences of VH and VL regions of exemplary antibodiesfor targets useful for treating disease, e.g., for treating cancer. Inan embodiment, the invention provides a DVD comprising at least two ofthe VH and/or VL regions listed in Table 2, in any orientation.

TABLE 2 List of Amino Acid Sequences of VH and VL regions of Antibodies for Generating DVD-Igs SEQ ABT ID Unique Protein Sequence No. ID region1234567890123456789012345678901234567890 28 AB001VH CD20QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS A 29 AB001VL CD20QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAE DAATYYCQQWTSNPPTFGGGTKLEIKR 30AB003VH EGFR QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR (1^(st) seq)QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS 31 AB003VL EGFRDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP (1^(st) seq)GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGTKVEIKR 32AB010VH IGF1,2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS 33 AB010VL IGF1,2QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCETWDTSLSAGRVFGGGTKLTVLG 34 AB017VH TNFEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS S 35 AB017VL TNFDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQGTKVEIKR 36AB020VH NGF QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVS S 37 AB020VL NGFDIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQP EDIATYYCQQEHTLPYTFGQGTKLEIKR 38AB032VH IL-1b QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS 39 AB032VL IL-1bEIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEA EDAAAYYCHQSSSLPFTFGPGTKVDIKR 40AB033VH EGFR QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS (2^(nd) seq)PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA 41 AB033VL EGFRDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT (2^(nd) seq)NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVES EDIADYYCQQNNNWPTTFGAGTKLELKR 42AB040VH IL-6 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSRLTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLT VSS 43 AB040VL IL-6QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAE DAATYYCQQWSGYPYTFGGGTKLEIKR 44AB043VH  Abeta EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQA (seq. 1)PGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 45 AB043VL  AbetaDVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNW (seq. 1)LLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKR 46 AB044VH  AbetaEVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQA (seq. 2)PGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 47 AB044VL  AbetaDVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEW (seq. 2)YLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKR 48 AB045VH Abeta EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQA (seq. 3) PGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS 49 AB045VL  AbetaDIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHW (seq. 3) YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKR 50 AB046VH IL-18EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVS S 51 AB046VL IL-18EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFGQGTRLEIKR52 AB052VH S1P EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCARGGFYGSTIWFDFWGQGTMVTVS S 53 AB052VL S1PETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLLISEGNILRPGVPSRFSSSGYGTDFTLTISKLQP EDFATYYCLQSDNLPFTFGQGTKLEIKR 54AB059VH RGMa EVQLVESGGGLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSS 55 AB059VL RGMaDVVLTQTPVSLSVTLGDQASMSCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIKR 56 AB060VH RAGEQIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGYINTNTGESIYSEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSRMVTAYGMDYWGQGTSVTVSS 57 AB060VL RAGEDIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQRPGQSPKLLIFSASNRYTGVPDRFTGSGSGTDFTLTLSNMQP EDLADYFCQQYSSYPLTFGVGTKLELKR

Detailed description of specific DVD-Ig molecules capable of bindingspecific targets, and methods of making the same, is provided in theExamples section below.

C. Production of DVD Proteins

Binding proteins of the present invention may be produced by any of anumber of techniques known in the art. For example, expression from hostcells, wherein expression vector(s) encoding the DVD heavy and DVD lightchains is (are) transfected into a host cell by standard techniques. Thevarious forms of the term “transfection” are intended to encompass awide variety of techniques commonly used for the introduction ofexogenous DNA into a prokaryotic or eukaryotic host cell, e.g.,electroporation, calcium-phosphate precipitation, DEAE-dextrantransfection and the like. Although it is possible to express the DVDproteins of the invention in either prokaryotic or eukaryotic hostcells, DVD proteins are expressed in eukaryotic cells, for example,mammalian host cells, because such eukaryotic cells (and in particularmammalian cells) are more likely than prokaryotic cells to assemble andsecrete a properly folded and immunologically active DVD protein.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufinan and P.A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells, SP2 and PER.C6 cells. Whenrecombinant expression vectors encoding DVD proteins are introduced intomammalian host cells, the DVD proteins are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe DVD proteins in the host cells or secretion of the DVD proteins intothe culture medium in which the host cells are grown. DVD proteins canbe recovered from the culture medium using standard protein purificationmethods.

In an exemplary system for recombinant expression of DVD proteins of theinvention, a recombinant expression vector encoding both the DVD heavychain and the DVD light chain is introduced into dhfr-CHO cells bycalcium phosphate-mediated transfection. Within the recombinantexpression vector, the DVD heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the DVD heavy and lightchains and intact DVD protein is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the DVD protein fromthe culture medium. Still further the invention provides a method ofsynthesizing a DVD protein of the invention by culturing a host cell ofthe invention in a suitable culture medium until a DVD protein of theinvention is synthesized. The method can further comprise isolating theDVD protein from the culture medium.

An important feature of DVD-Ig is that it can be produced and purifiedin a similar way as a conventional antibody. The production of DVD-Igresults in a homogeneous, single major product with desireddual-specific activity, without any sequence modification of theconstant region or chemical modifications of any kind. Other previouslydescribed methods to generate “bi-specific”, “multi-specific”, and“multi-specific multivalent” full length binding proteins do not lead toa single primary product but instead lead to the intracellular orsecreted production of a mixture of assembled inactive, mono-specific,multi-specific, multivalent, full length binding proteins, andmultivalent full length binding proteins with combination of differentbinding sites. As an example, based on the design described by Millerand Presta (PCT publication WO2001/077342(A1), there are 16 possiblecombinations of heavy and light chains. Consequently only 6.25% ofprotein is likely to be in the desired active form, and not as a singlemajor product or single primary product compared to the other 15possible combinations. Separation of the desired, fully active forms ofthe protein from inactive and partially active forms of the proteinusing standard chromatography techniques, typically used in large scalemanufacturing, is yet to be demonstrated.

Surprisingly the design of the “dual-specific multivalent full lengthbinding proteins” of the present invention leads to a dual variabledomain light chain and a dual variable domain heavy chain which assembleprimarily to the desired “dual-specific multivalent full length bindingproteins”.

At least 50%, at least 75% and at least 90% of the assembled, andexpressed dual variable domain immunoglobulin molecules are the desireddual-specific tetravalent protein. This aspect of the inventionparticularly enhances the commercial utility of the invention.Therefore, the present invention includes a method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a single primary product of a “dual-specifictetravalent full length binding protein”.

The present invention provides a methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a “primary product” of a “dual-specific tetravalent fulllength binding protein”, where the “primary product” is more than 50% ofall assembled protein, comprising a dual variable domain light chain anda dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 75% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 90% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

II. Derivatized DVD Binding Proteins: One embodiment provides a labeledbinding protein wherein the binding protein of the invention isderivatized or linked to another functional molecule (e.g., anotherpeptide or protein). For example, a labeled binding protein of theinvention can be derived by functionally linking an binding protein ofthe invention (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetectable agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate association of the binding proteinwith another molecule (such as a streptavidin core region or apolyhistidine tag).

Useful detectable agents with which a binding protein of the inventionmay be derivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. A binding protein may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When a binding protein is derivatized with adetectable enzyme, it is detected by adding additional reagents that theenzyme uses to produce a detectable reaction product. For example, whenthe detectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. a binding protein may also be derivatizedwith biotin, and detected through indirect measurement of avidin orstreptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein and formulations and compositions comprising such crystals. Inone embodiment the crystallized binding protein has a greater half-lifein vivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingto methods known in the art and as disclosed in WO 02072636,incorporated herein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have importanteffect on the effector function of the Fc domain, with minimal effect onantigen binding or half-life of the antibody (R. Jefferis, Biotechnol.Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Co,M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result inincreased affinity for the antigen (Wallick, S. C., et al., Exp. Med.(1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity but haveincreased or decreased binding activity are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified binding protein of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues(see Kanda, Yutaka et al., Journal of Biotechnology (2007), 130(3),300-310.) or an antibody having increased bisecting GlcNAc structures.Such altered glycosylation patterns have been demonstrated to increasethe ADCC ability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (eg.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. In an embodiment, the glycosylatedbinding protein comprises glycosyl residues such that the glycosylationpattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner maychoose a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S patent applications20040018590 and 20020137134 and PCT publication WO2005100584 A2).

In addition to the binding proteins, the present invention is alsodirected to anti-idiotypic (anti-Id) antibodies specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. It is readily apparent that it may be easier to generateanti-idiotypic antibodies to the two or more parent antibodiesincorporated into a DVD-Ig molecule; and confirm binding studies bymethods well recognized in the art (e.g., BIAcore, ELISA) to verify thatanti-idiotypic antibodies specific for the idiotype of each parentantibody also recognize the idiotype (e.g., antigen binding site) in thecontext of the DVD-Ig. The anti-idiotypic antibodies specific for eachof the two or more antigen binding sites of a DVD-Ig provide idealreagents to measure DVD-Ig concentrations of a human DVD-Ig in patrientserum; DVD-Ig concentration assays can be established using a “sandwichassay ELISA format” with an antibody to a first antigen binding regionscoated on the solid phase (e.g., BIAcore chip, ELISA plate etc.), rinsedwith rinsing buffer, incubation with the serum sample, another rinsingstep and ultimately incubation with another anti-idiotypic antibody tothe another antigen binding site, itself labeled with an enzyme forquantitation of the binding reaction. In an embodiment, for a DVD-Igwith more than two different binding sites, anti-idiotypic antibodies tothe two outermost binding sites (most distal and proximal from theconstant region) will not only help in determining the DVD-Igconcentration in human serum but also document the integrity of themolecule in vivo. Each anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns. In anembodiment, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

III. Uses of DVD-Ig

Given their ability to bind to two or more antigens the binding proteinsof the invention can be used to detect the antigens (e.g., in abiological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The DVD-Ig isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

In an embodiment, the binding proteins of the invention are capable ofneutralizing the activity of the antigens both in vitro and in vivo.Accordingly, such DVD-Igs can be used to inhibit antigen activity, e.g.,in a cell culture containing the antigens, in human subjects or in othermammalian subjects having the antigens with which a binding protein ofthe invention cross-reacts. In another embodiment, the inventionprovides a method for reducing antigen activity in a subject sufferingfrom a disease or disorder in which the antigen activity is detrimental.A binding protein of the invention can be administered to a humansubject for therapeutic purposes.

As used herein, the term “a disorder in which antigen activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of the antigen in a subject suffering from thedisorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a disorder inwhich antigen activity is detrimental is a disorder in which reductionof antigen activity is expected to alleviate the symptoms and/orprogression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of the antigen in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of antigen in serum, plasma, synovialfluid, etc. of the subject). Non-limiting examples of disorders that canbe treated with the binding proteins of the invention include thosedisorders discussed below and in the section pertaining topharmaceutical compositions of the antibodies of the invention.

The DVD-Igs of the invention may bind one antigen or multiple antigens.Such antigens include, but are not limited to, the targets listed in thefollowing databases, which databases are incorporated herein byreference. These target databases include those listings:

-   Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);-   Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/,    http://www.copewitheytokines.de/cope.cgi, and-   http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-u.ac.jp/CFC/indexR.html);-   Chemokines    (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);-   Chemokine receptors and GPCRs    (http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html,    http://www.gper.org/7tm/);-   Olfactory Receptors (http://s ens elab.med.yale edu/sens    elab/ORDB/default.asp);-   Receptors (http://www.iuphar-db.org/iuphar-rd/list/index.htm);-   Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi);-   Secreted proteins as potential antibody targets    (http://spd.cbi.pku.edu.cn/);-   Protein kinases (http://spd.cbi.pku.edu.cn/), and-   Human CD markers    (http://content.labvelocity.com/tools/6/1226/CDtable_final    Jocked.pdf) and (Zola H, 2005 CD molecules 2005: human cell    differentiation molecules Blood, 106:3123-6).

DVD-Igs are useful as therapeutic agents to simultaneously block twodifferent targets to enhance efficacy/safety and/or increase patientcoverage. Such targets may include soluble targets (TNF) and cellsurface receptor targets (VEGFR and EGFR). It can also be used to induceredirected cytotoxicity between tumor cells and T cells (Her2 and CD3)for cancer therapy, or between autoreactive cell and effector cells forautoimmune disease or transplantation, or between any target cell andeffector cell to eliminate disease-causing cells in any given disease.

In addition, DVD-Ig can be used to trigger receptor clustering andactivation when it is designed to target two different epitopes on thesame receptor. This may have benefit in making agonistic andantagonistic anti-GPCR therapeutics. In this case, DVD-Ig can be used totarget two different epitopes (including epitopes on both the loopregions and the extracellular domain) on one cell forclustering/signaling (two cell surface molecules) or signaling (on onemolecule). Similarly, a DVD-Ig molecule can be designed to triger CTLA-4ligation, and a negative signal by targeting two different epitopes (or2 copies of the same epitope) of CTLA-4 extracellular domain, leading todown regulation of the immune response. CTLA-4 is a clinically validatedtarget for therapeutic treatment of a number of immunological disorders.CTLA-4/B7 interactions negatively regulate T cell activation byattenuating cell cycle progression, IL-2 production, and proliferationof T cells following activation, and CTLA-4 (CD152) engagement candown-regulate T cell activation and promote the induction of immunetolerance. However, the strategy of attenuating T cell activation byagonistic antibody engagement of CTLA-4 has been unsuccessful sinceCTLA-4 activation requires ligation. The molecular interaction ofCTLA-4/B7 is in “skewed zipper” arrays, as demonstrated by crystalstructural analysis (Stamper 2001 Nature 410:608). However none of thecurrently available CTLA-4 binding reagents have ligation properties,including anti-CTLA-4 mAbs. There have been several attempts to addressthis issue. In one case, a cell member-bound single chain antibody wasgenerated, and significantly inhibited allogeneic rejection in mice(Hwang 2002 JI 169:633). In a separate case, artificial APCsurface-linked single-chain antibody to CTLA-4 was generated anddemonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433).In both cases, CTLA-4 ligation was achieved by closely localizedmember-bound antibodies in artificial systems. While these experimentsprovide proof-of-concept for immune down-regulation by triggering CTLA-4negative signaling, the reagents used in these reports are not suitablefor therapeutic use. To this end, CTLA-4 ligation may be achieved byusing a DVD-Ig molecule, which target two different epitopes (or 2copies of the same epitope) of CTLA-4 extracellular domain. Therationale is that the distance spanning two binding sites of an IgG,approximately 150-170A, is too large for active ligation of CTLA-4(30-50 Å between 2 CTLA-4 homodimer). However the distance between thetwo binding sites on DVD-Ig (one arm) is much shorter, also in the rangeof 30-50 Å, allowing proper ligation of CTLA-4.

Similarly, DVD-Ig can target two different members of a cell surfacereceptor complex (e.g., IL-12R alpha and beta). Furthermore, DVD-Ig cantarget CR1 and a soluble protein/pathogen to drive rapid clearance ofthe target soluble protein/pathogen.

Additionally, DVD-Igs of the invention can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand a intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). DVD-Ig can also serve as a carrier protein todeliver an antigen to a specific location via binding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, DVD-Ig can be designed to eitherbe physically linked to medical devices implanted into patients ortarget these medical devices (see Burke, Sandra E.; Kuntz, Richard E.;Schwartz, Lewis B., Zotarolimus eluting stents. Advanced Drug DeliveryReviews (2006), 58(3), 437-446; Surface coatings for biologicalactivation and functionalization of medical devices, Hildebrand, H. F.;Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F., Surfaceand Coatings Technology (2006), 200(22-23), 6318-6324; Drug/devicecombinations for local drug therapies and infection prophylaxis, Wu,Peng; Grainger, David W., Biomaterials (2006), 27(11), 2450-2467;Mediation of the cytokine network in the implantation of orthopedicdevices., Marques, A. P.; Hunt, J. A.; Reis, Rui L., BiodegradableSystems in Tissue Engineering and Regenerative Medicine (2005),377-397). Briefly, directing appropriate types of cell to the site ofmedical implant may promote healing and restoring normal tissuefunction. Alternatively, inhibition of mediators (including but notlimited to cytokines), released upon device implantation by a DVDcoupled to or target to a device is also provided. For example, Stentshave been used for years in interventional cardiology to clear blockedarteries and to improve the flow of blood to the heart muscle. However,traditional bare metal stents have been known to cause restenosis(re-narrowing of the artery in a treated area) in some patients and canlead to blood clots. Recently, an anti-CD34 antibody coated stent hasbeen described which reduced restenosis and prevents blood clots fromoccurring by capturing endothelial progenitor cells (EPC) circulatingthroughout the blood. Endothelial cells are cells that line bloodvessels, allowing blood to flow smoothly. The EPCs adhere to the hardsurface of the stent forming a smooth layer that not only promoteshealing but prevents restenosis and blood clots, complicationspreviously associated with the use of stents (Aoji et al. 2005 J Am CollCardiol. 45(10):1574-9). In addition to improving outcomes for patientsrequiring stents, there are also implications for patients requiringcardiovascular bypass surgery. For example, a prosthetic vascularconduit (artificial artery) coated with anti-EPC antibodies wouldeliminate the need to use arteries from patients legs or arms for bypasssurgery grafts. This would reduce surgery and anesthesia times, which inturn will reduce coronary surgery deaths. DVD-Ig are designed in such away that it binds to a cell surface marker (such as CD34) as well as aprotein (or an epitope of any kind, including but not limited toproteins, lipids and polysaccharides) that has been coated on theimplanted device to facilitate the cell recruitment. Such approaches canalso be applied to other medical implants in general. Alternatively,DVD-Igs can be coated on medical devices and upon implantation andreleasing all DVDs from the device (or any other need which may requireadditional fresh DVD-Ig, including aging and denaturation of the alreadyloaded DVD-Ig) the device could be reloaded by systemic administrationof fresh DVD-Ig to the patient, where the DVD-Ig is designed to binds toa target of interest (a cytokine, a cell surface marker (such as CD34)etc.) with one set of binding sites and to a target coated on the device(including a protein, an epitope of any kind, including but not limitedto lipids, polysaccharides and polymers) with the other. This technologyhas the advantage of extending the usefulness of coated implants.

A. Use of DVD-Igs in Various Diseases

DVD-Ig molecules of the invention are also useful as therapeuticmolecules to treat various diseases. Such DVD molecules may bind one ormore targets involved in a specific disease. Examples of such targets invarious diseases are described below.

1. Human Autoimmune and Inflammatory Response

Many proteins have been implicated in general autoimmune andinflammatory responses, including C5, CCL1 (1-309), CCL11 (eotaxin),CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23(MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a),CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10(IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2,CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13(mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1,IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10,IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF,SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5,IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3,C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4,TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G,CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A,GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11,CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2,BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3,DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1,IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R,IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11,IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15,IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA,LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,TGFB1I1, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). Inone aspect, DVD-Igs capable of binding one or more of the targets listedherein are provided.

2. Asthma

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. It is now widely accepted that airway inflammation is thekey factor underlying the pathogenesis of asthma, involving a complexinterplay of inflammatory cells such as T cells, B cells, eosinophils,mast cells and macrophages, and of their secreted mediators includingcytokines and chemokines. Corticosteroids are the most importantanti-inflammatory treatment for asthma today, however their mechanism ofaction is non-specific and safety concerns exist, especially in thejuvenile patient population. The development of more specific andtargeted therapies is therefore warranted. There is increasing evidencethat IL-13 in mice mimics many of the features of asthma, including AHR,mucus hypersecretion and airway fibrosis, independently of eosinophilicinflammation (Finotto et al., International Immunology (2005), 17(8),993-1007; Padilla et al., Journal of Immunology (2005), 174(12),8097-8105).

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. The development ofanti-IL-13 mAb therapy to reduce the effects of IL-13 in the lung is anexciting new approach that offers considerable promise as a noveltreatment for asthma. However other mediators of differentialimmunological pathways are also involved in asthma pathogenesis, andblocking these mediators, in addition to IL-13, may offer additionaltherapeutic benefit. Such target pairs include, but are not limited to,IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α(TNF-α). TNF-α may amplify the inflammatory response in asthma and maybe linked to disease severity (McDonnell, et al., Progress inRespiratory Research (2001), 31(New Drugs for Asthma, Allergy and COPD),247-250.). This suggests that blocking both IL-13 and TNF-α may havebeneficial effects, particularly in severe airway disease. In anotherembodiment the DVD-Ig of the invention binds the targets IL-13 and TNFαand is used for treating asthma.

Animal models such as OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various DVD-Ig molecules to treatasthma Animal models for studying asthma are disclosed in Coffman, etal., Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd,et al., Advances in Immunology (2001), 77, 263-295; Boyce et al.,Journal of Experimental Medicine (2005), 201(12), 1869-1873; andSnibson, et al., Journal of the British Society for Allergy and ClinicalImmunology (2005), 35(2), 146-52. In addition to routine safetyassessments of these target pairs specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Hart et al., Journal of Allergy and Clinical Immunology (2001),108(2), 250-257).

Based on the rationale disclosed herein and using the same evaluationmodel for efficacy and safety other pairs of targets that DVD-Igmolecules can bind and be useful to treat asthma may be determined. Inan embodiment, such targets include, but are not limited to, IL-13 andIL-1beta, since IL-1beta is also implicated in inflammatory response inasthma; IL-13 and cytokines and chemokines that are involved ininflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5;IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; and IL-13 and ADAMS. The present invention also providesDVD-Igs capable of binding one or more targets involved in asthmaselected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3(GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors,IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A,IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA,IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1,TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18,CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3,JAK1, JAK3, STATE, TBX21, TGFB1, TNF, TNFSF6, YY1, CYSLTR1, FCER1A,FCER2, LTB4R, TB4R2, LTBR, and Chitinase.

3. Rheumatoid Arthritis

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines including TNF, chemokines, andgrowth factors are expressed in diseased joints. Systemic administrationof anti-TNF antibody or sTNFR fusion protein to mouse models of RA wasshown to be anti-inflammatory and joint protective. Clinicalinvestigations in which the activity of TNF in RA patients was blockedwith intravenously administered infliximab (Harriman G, Harper L K,Schaible T F. 1999 Summary of clinical trials in rheumatoid arthritisusing infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl1:161-4), a chimeric anti-TNF mAb, has provided evidence that TNFregulates IL-6, IL-8, MCP-1, and VEGF production, recruitment of immuneand inflammatory cells into joints, angiogenesis, and reduction of bloodlevels of matrix metalloproteinases⁻¹ and -3. A better understanding ofthe inflammatory pathway in rheumatoid arthritis has led toidentification of other therapeutic targets involved in rheumatoidarthritis. Promising treatments such as interleukin-6 antagonists (IL-6receptor antibody MRA, developed by Chugai, Roche (see Nishimoto,Norihiro et al., Arthritis & Rheumatism (2004), 50(6), 1761-1769),CTLA4Ig (abatacept, Genovese Mc et al 2005 Abatacept for rheumatoidarthritis refractory to tumor necrosis factor alpha inhibition. N Engl JMed. 353:1114-23.), and anti-B cell therapy (rituximab, Okamoto H,Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med.351:1909) have already been tested in randomized controlled trials overthe past year. Other cytokines have been identified and have been shownto be of benefit in animal models, including interleukin-15 (therapeuticantibody HuMax-IL_(—)15, AMG 714 see Baslund, Bo et al., Arthritis &Rheumatism (2005), 52(9), 2686-2692), interleukin-17, andinterleukin-18, and clinical trials of these agents are currently underway. Dual-specific antibody therapy, combining anti-TNF and anothermediator, has great potential in enhancing clinical efficacy and/orpatient coverage. For example, blocking both TNF and VEGF canpotentially eradicate inflammation and angiogenesis, both of which areinvolved in pathophysiology of RA. Blocking other pairs of targetsinvolved in RA including, but not limited to, TNF and IL-18; TNF andIL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; andTNF and IL-15 with specific DVD Igs is also contemplated. In addition toroutine safety assessments of these target pairs, specific tests for thedegree of immunosuppression may be warranted and helpful in selectingthe best target pairs (see Luster et al., Toxicology (1994), 92(1-3),229-43; Descotes, et al., Developments in biological standardization(1992), 77 99-102; Hart et al., Journal of Allergy and ClinicalImmunology (2001), 108(2), 250-257). Whether a DVD Ig molecule will beuseful for the treatment of rheumatoid arthritis can be assessed usingpre-clinical animal RA models such as the collagen-induced arthritismouse model. Other useful models are also well known in the art (seeBrand DD., Comp Med. (2005) 55(2):114-22). Based on the cross-reactivityof the parental antibodies for human and mouse othologues (e.g.,reactivity for human and mouse TNF, human and mouse IL-15 etc.)validation studies in the mouse CIA model may be conducted with “matchedsurrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based ontwo (or more) mouse target specific antibodies may be matched to theextent possible to the characteristics of the parental human orhumanized antibodies used for human DVD-Ig construction (similaraffinity, similar neutralization potency, similar half-life etc.).

4. SLE

The immunopathogenic hallmark of SLE is the polyclonal B cellactivation, which leads to hyperglobulinemia, autoantibody productionand immune complex formation. The fundamental abnormality appears to bethe failure of T cells to suppress the forbidden B cell clones due togeneralized T cell dysregulation. In addition, B and T-cell interactionis facilitated by several cytokines such as IL-1β as well asco-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4,which initiate the second signal. These interactions together withimpaired phagocytic clearance of immune complexes and apoptoticmaterial, perpetuate the immune response with resultant tissue injury.The following targets may be involved in SLE and can potentially be usedfor DVD-Ig approach for therapeutic intervention: B cell targetedtherapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C,CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatory signals: CTLA4 orB7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complementinactivation: C5; Cytokine modulation: the key principle is that the netbiologic response in any tissue is the result of a balance between locallevels of proinflammatory or anti-inflammatory cytokines (see Sfikakis PP et al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be aTh-2 driven disease with documented elevations in serum IL-4, IL-6,IL-10. DVD Igs capable of binding one or more targets selected from thegroup consisting of IL-4, IL-6, IL-10, IFN-α, and TNF-α are alsocontemplated. Combination of targets discussed herein will enhancetherapeutic efficacy for SLE which can be tested in a number of lupuspreclinical models (see Peng S L (2004) Methods Mol Med.; 102:227-72).Based on the cross-reactivity of the parental antibodies for human andmouse othologues (e.g., reactivity for human and mouse CD20, human andmouse Interferon alpha etc.) validation studies in a mouse lupus modelmay be conducted with “matched surrogate antibody” derived DVD-Igmolecules; briefly, a DVD-Ig based two (or more) mouse target specificantibodies may be matched to the extent possible to the characteristicsof the parental human or humanized antibodies used for human DVD-Igconstruction (similar affinity, similar neutralization potency, similarhalf-life etc.).

5. Multiple Sclerosis

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology Immunologic destruction of myelin basicprotein (MBP) throughout the nervous system is the major pathology ofmultiple sclerosis. MS is a disease of complex pathologies, whichinvolves infiltration by CD4+ and CD8+ T cells and of response withinthe central nervous system. Expression in the CNS of cytokines, reactivenitrogen species and costimulator molecules have all been described inMS. Of major consideration are immunological mechanisms that contributeto the development of autoimmunity. In particular, antigen expression,cytokine and leukocyte interactions, and regulatory T-cells, which helpbalance/modulate other T-cells such as Th1 and Th2 cells, are importantareas for therapeutic target identification.

IL-12 is a proinflammatory cytokine that is produced by APC and promotesdifferentiation of Th1 effector cells. IL-12 is produced in thedeveloping lesions of patients with MS as well as in EAE-affectedanimals. Previously it was shown that interference in IL-12 pathwayseffectively prevents EAE in rodents, and that in vivo neutralization ofIL-12p40 using a anti-IL-12 mAb has beneficial effects in themyelin-induced EAE model in common marmosets.

TWEAK is a member of the TNF family, constitutively expressed in thecentral nervous system (CNS), with pro-inflammatory, proliferative orapoptotic effects depending upon cell types. Its receptor, Fn14, isexpressed in CNS by endothelial cells, reactive astrocytes and neurons.TWEAK and Fn14 mRNA expression increased in spinal cord duringexperimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibodytreatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE inC57BL/6 mice resulted in a reduction of disease severity and leukocyteinfiltration when mice were treated after the priming phase.

One aspect of the invention pertains to DVD Ig molecules capable ofbinding one or more, for example two, targets selected from the groupconsisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF,VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. Anembodiment includes a dual-specific anti-IL-12/TWEAK DVD Ig as atherapeutic agent beneficial for the treatment of MS.

Several animal models for assessing the usefulness of the DVD moleculesto treat MS are known in the art (see Steinman L, et al., (2005) TrendsImmunol. 26(11):565-71; Lublin F D., et al., (1985) Springer SeminImmunopathol.8(3):197-208; Genain C P, et al., (1997) J Mol Med.75(3):187-97; Tuohy V K, et al., (1999) J Exp Med. 189(7):1033-42; OwensT, et al., (1995) Neurol Clin.13(1):51-73; and ^(s)t Hart B A, et al.,(2005) J Immunol 175(7):4761-8. Based on the cross-reactivity of theparental antibodies for human and animal species othologues (e.g.,reactivity for human and mouse IL-12, human and mouse TWEAK etc.)validation studies in the mouse EAE model may be conducted with “matchedsurrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based onto (or more) mouse target specific antibodies may be matched to theextent possible to the characteristics of the parental human orhumanized antibodies used for human DVD-Ig construction (similaraffinity, similar neutralization potency, similar half-life etc.). Thesame concept applies to animal models in other non-rodent species, wherea “matched surrogate antibody” derived DVD-Ig would be selected for theanticipated pharmacology and possibly safety studies. In addition toroutine safety assessments of these target pairs specific tests for thedegree of immunosuppression may be warranted and helpful in selectingthe best target pairs (see Luster et al., Toxicology (1994), 92(1-3),229-43; Descotes, et al., Developments in biological standardization(1992), 77 99-102; Jones R. 2000 Rovelizumab (ICOS Corp).IDrugs.3(4):442-6).

MS is however not only an imunologic disease but has a very importantneurodegenerative component. Disease progression in MS is due tocumulative loss and damage of axons and the final disease scores of thepatients are determined by these neurodegenerative processes (CompstonA. & Coles A. (2008) Lancet 372: 1502-1517; Trapp B D. & Nave K A.(2008) Annu. Rev. Neuroscience 31: 247-269). Several mechanisms mightaccount for axonal damage in MS. Excessive release of theneurotransmitter glutamate with associated calcium-mediatedneurotoxicity, nitric-oxide release and subsequent axon damage, loss ofneurotrophic support, massive accumulation of repulsive or axon growthinhibitory molecules like RGM A, NOGO A, Semaphorins, Ephrins, maycontribute to axon-directed neurodegeneration and loss of successfulaxon regeneration. Targeting in a single DVD Ig molecule neutralizingactivities directed against components like RGM A, NOGO A, Semaphorins,Ephrins with neutralizing activities directed against pro-inflammatorycytokines like IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF,VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2would enable the simultaneous focus on inflammation andneuroregeneration, a goal not yet achieved by any of the currenttherapeutic MS principles. Stimulating neuroregeneration can compensatethe functional impairments caused by the massive axonalneurodegeneration observed in MS, making recovery of lost cerebralfunctions possible.

6. Sepsis

The pathophysiology of sepsis is initiated by the outer membranecomponents of both gram-negative organisms (lipopolysaccharide [LPS],lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid,peptidoglycan). These outer membrane components are able to bind to theCD14 receptor on the surface of monocytes. By virtue of the recentlydescribed toll-like receptors, a signal is then transmitted to the cell,leading to the eventual production of the proinflammatory cytokinestumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1).Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokines,especially tumor necrosis factor (TNF) and interleukin (IL-1), have beenshown to be critical mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity.

The treatment of sepsis and septic shock remains a clinical conundrum,and recent prospective trials with biological response modifiers (i.e.anti-TNF, anti-MIF) aimed at the inflammatory response have shown onlymodest clinical benefit. Recently, interest has shifted toward therapiesaimed at reversing the accompanying periods of immune suppression.Studies in experimental animals and critically ill patients havedemonstrated that increased apoptosis of lymphoid organs and someparenchymal tissues contribute to this immune suppression, anergy, andorgan system dysfunction. During sepsis syndromes, lymphocyte apoptosiscan be triggered by the absence of IL-2 or by the release ofglucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumornecrosis factor alpha or Fas ligand. Apoptosis proceeds viaauto-activation of cytosolic and/or mitochondrial caspases, which can beinfluenced by the pro- and anti-apoptotic members of the Bcl-2 family.In experimental animals, not only can treatment with inhibitors ofapoptosis prevent lymphoid cell apoptosis; it may also improve outcome.Although clinical trials with anti-apoptotic agents remain distant duein large part to technical difficulties associated with theiradministration and tissue targeting, inhibition of lymphocyte apoptosisrepresents an attractive therapeutic target for the septic patient.Likewise, a dual-specific agent targeting both inflammatory mediator anda apoptotic mediator, may have added benefit. One aspect of theinvention pertains to DVD Igs capable of binding one or more targetsinvolved in sepsis, in an embodiment two targets, selected from thegroup consisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL,LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1,CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3, CCR3, IL1RN, MIF,NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1,SERPINE1, and TREM1. The efficacy of such DVD Igs for sepsis can beassessed in preclinical animal models known in the art (see Buras J A,et al., (2005) Nat Rev Drug Discov. 4(10):854-65 and Calandra T, et al.,(2000) Nat Med. 6(2):164-70).

7. Neurological Disorders 7.1. Neurodegenerative Diseases

Neurodegenerative diseases are either chronic in which case they areusually age-dependent or acute (e.g., stroke, traumatic brain injury,spinal cord injury, etc.). They are characterized by progressive loss ofneuronal functions (neuronal cell death, axon loss, neuritic dystrophy,demyelination), loss of mobility and loss of memory. Emerging knowledgeof the mechanisms underlying chronic neurodegenerative diseases (e.g.,Alzheimer's disease disease) show a complex etiology and a variety offactors have been recognized to contribute to their development andprogression e.g., age, glycemic status, amyloid production andmultimerization, accumulation of advanced glycation-end products (AGE)which bind to their receptor RAGE (receptor for AGE), increased brainoxidative stress, decreased cerebral blood flow, neuroinflammationincluding release of inflammatory cytokines and chemokines, neuronaldysfunction and microglial activation. Thus these chronicneurodegenerative diseases represent a complex interaction betweenmultiple cell types and mediators. Treatment strategies for suchdiseases are limited and mostly constitute either blocking inflammatoryprocesses with non-specific anti-inflammatory agents (e.g.,corticosteroids, COX inhibitors) or agents to prevent neuron loss and/orsynaptic functions. These treatments fail to stop disease progression.Recent studies suggest that more targeted therapies such as antibodiesto soluble A-b peptide (including the A-b oligomeric forms) can not onlyhelp stop disease progression but may help maintain memory and othercognitive functions as well. These preliminary observations suggest thatspecific therapies targeting more than one disease mediator (e.g., A-band a pro-inflammatory cytokine such as TNF) may provide even bettertherapeutic efficacy for chronic neurodegenerative diseases thanobserved with targeting a single disease mechanism (e.g., soluble A-balone) (see C.E. Shepherd, et al, Neurobiol Aging. 2005 Oct. 24; NelsonR B., Curr Pharm Des. 2005; 11:3335; William L. Klein.; Neurochem Int.2002; 41:345; Michelle C Janelsins, et al., J Neuroinflammation. 2005;2:23; Soloman B., Curr Alzheimer Res. 2004; 1:149; Igor Klyubin, et al.,Nat Med. 2005; 11:556-61; Arancio 0, et al., EMBO Journal (2004) 1-10;Bornemann K D, et al., Am J Pathol. 2001; 158:63; Deane R, et al., NatMed. 2003; 9:907-13; and Eliezer Masliah, et al., Neuron. 2005; 46:857).

The DVD-Ig molecules of the invention can bind one or more targetsinvolved in Chronic neurodegenerative diseases such as Alzheimers. Suchtargets include, but are not limited to, any mediator, soluble or cellsurface, implicated in AD pathogenesis e.g AGE (S100 A, amphoterin),pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MCP 1),molecules that inhibit nerve regeneration (e.g., Nogo, RGM A), moleculesthat enhance neurite growth (neurotrophins) and molecules that canmediate transport at the blood brain barrier (e.g., transferrinreceptor, insulin receptor or RAGE). The efficacy of DVD-Ig moleculescan be validated in pre-clinical animal models such as the transgenicmice that over-express amyloid precursor protein or RAGE and developAlzheimer's disease-like symptoms. In addition, DVD-Ig molecules can beconstructed and tested for efficacy in the animal models and the besttherapeutic DVD-Ig can be selected for testing in human patients. DVD-Igmolecules can also be employed for treatment of other neurodegenerativediseases such as Parkinson's disease. Alpha-Synuclein is involved inParkinson's pathology. A DVD-Ig capable of targeting alpha-synuclein andinflammatory mediators such as TNF, IL-1, MCP-1 can prove effectivetherapy for Parkinson's disease and are contemplated in the invention.

Alternatively a DVD-Ig capable of targeting alpha-synuclein and RGM Acould not only halt the pathologic progress in the substantia nigra ofParkinson disease patients but could also result in regenerative growthof damaged neurites because RGM A has been recently shown to be stronglyupregulated in this area in PD patients (Bossers K. et al. (2009) BrainPathol. 19: 91-107).

7.2 Neuronal Regeneration and Spinal Cord Injury

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g., cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. These primary and secondary mechanisms in SCI are verysimilar to those in brain injury caused by other means, e.g., trauma andstroke. No satisfying treatment exists and high dose bolus injection ofmethylprednisolone (MP) is the only used therapy within a narrow timewindow of 8 h post injury. This treatment, however, is only intended toprevent secondary injury without causing any significant functionalrecovery. It is heavily critisized for the lack of unequivocal efficacyand severe adverse effects, like immunosuppression with subsequentinfections and severe histopathological muscle alterations. No otherdrugs, biologics or small molecules, stimulating the endogenousregenerative potential are approved, but promising treatment principlesand drug candidates have shown efficacy in animal models of SCI inrecent years and first promising clinical data have been presented justrecently. To a large extent the lack of functional recovery in human SCIis caused by factors inhibiting neurite growth, at lesion sites, in scartissue, in myelin as well as on injury-associated cells. Such factorsare the myelin-associated proteins NogoA, OMgp and MAG, RGM A, thescar-associated CSPG (Chondroitin Sulfate Proteoglycans) and inhibitoryfactors on reactive astrocytes (some semaphorins and ephrins). However,at the lesion site not only growth inhibitory molecules are found butalso neurite growth stimulating factors like neurotrophins, laminin, L1and others. This ensemble of neurite growth inhibitory and growthpromoting molecules may explain that blocking single factors, like NogoAor RGM A, resulted in significant functional recovery in rodent SCImodels, because a reduction of the inhibitory influences could shift thebalance from growth inhibition to growth promotion. However, recoveriesobserved with blocking a single neurite outgrowth inhibitory moleculewere not complete. To achieve faster and more pronounced recoverieseither blocking two neurite outgrowth inhibitory molecules e.g Nogo andRGM A, or blocking an neurite outgrowth inhibitory molecule andenhancing functions of a neurite outgrowth enhancing molecule e.g Nogoand neurotrophins, or blocking a neurite outgrowth inhibitory moleclulee.g., Nogo and a pro-inflammatory molecule e.g., TNF, may be desirable(see McGee A W, et al., Trends Neurosci. 2003; 26:193; Marco Domeniconi,et al., J Neurol Sci. 2005; 233:43; Milan Makwanal, et al., FEBS J.2005; 272:2628; Barry J. Dickson, Science. 2002; 298:1959; Felicia YuHsuan Teng, et al., J Neurosci Res. 2005; 79:273; Tara Karnezis, et al.,Nature Neuroscience 2004; 7, 736; Gang Xu, et al., J. Neurochem.2004;91; 1018).

In one aspect, DVD-Igs capable of binding target pairs such as NgR andRGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B;RGM A and Semaphorin 3A; RGM A and Semaphorin 4; CSPGs and RGM A;aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-α; ABglobulomer-specific antibodies combined with antibodies promotingdendrite & axon sprouting are provided. Dendrite pathology and axondamage, or neuritic dystrophy are a very early sign of AD and it isknown that NOGO A restricts dendrite growth and that the other moleculesassociated with myelin and mentioned above e.g., RGM A, MAG, OMGp impairaxonal regrowth. One can combine such type of ab with any of theSCI-candidate (myelin-proteins) Ab. Other DVD-Ig targets may include anycombination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo,Lingo-Troy, Lingo-p75, MAG or Omgp. Additionally, targets may alsoinclude any mediator, soluble or cell surface, implicated in inhibitionof neurite e.g Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, solubleA-b, pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MIP 1a),molecules that inhibit nerve regeneration. The efficacy ofanti-nogo/anti-RGM A or similar DVD-Ig molecules can be validated inpre-clinical animal models of spinal cord injury. In addition, theseDVD-Ig molecules can be constructed and tested for efficacy in theanimal models and the best therapeutic DVD-Ig can be selected fortesting in human patients. In addition, DVD-Ig molecules can beconstructed that target two distinct ligand binding sites on a singlereceptor e.g., Nogo receptor which binds three ligand Nogo, OMGp, andMAG and RAGE that binds A-b and S100 A.

Furthermore, neurite outgrowth inhibitors e.g., nogo and nogo receptor,also play a role in preventing nerve regeneration in immunologicaldiseases like multiple sclerosis Inhibition of nogo-nogo receptorinteraction has been shown to enhance recovery in animal models ofmultiple sclerosis. Therefore, DVD-Ig molecules that can block thefunction of one immune mediator e.g., a cytokine like IL-12 and aneurite outgrowth inhibitor molecule e.g., nogo or RGM may offer fasterand greater efficacy than blocking either an immune or an neuriteoutgrowth inhibitor molecule alone.

In general, antibodies do not cross the blood brain barrier (BBB) in anefficient and relevant manner. However, in certain neurologic diseases,e.g., stroke, traumatic brain injury, multiple sclerosis, etc., the BBBmay be compromised and allows for increased penetration of DVD-Igs andantibodies into the brain. In other neurological conditions, where BBBleakage is not occurring, one may employ the targeting of endogenoustransport systems, including carrier-mediated transporters such asglucose and amino acid carriers and receptor-mediatedtranscytosis-mediating cell structures/receptors at the vascularendothelium of the BBB, thus enabling trans-BBB transport of the DVD-Ig.Structures at the BBB enabling such transport include but are notlimited to the insulin receptor, transferrin receptor, LRP and RAGE. Inaddition, strategies enable the use of DVD-Igs also as shuttles totransport potential drugs into the CNS including low molecular weightdrugs, nanoparticles and nucleic acids (Coloma M J, et al. (2000) PharmRes. 17(3):266-74; Boado R J, et al. (2007) Bioconjug. Chem.18(2):447-55).

DVD Igs capable of binding the following pairs of targets to treatneurological disease are contemplated: Abeta (seq. 1) and Abeta (seq.3); TNF and Abeta (seq. 2); IL-1b and Abeta (seq. 2); Abeta (seq. 1) andAbeta (seq. 2); IGF1,2 and Abeta (seq. 2); Abeta (seq. 2) and IL-18;IL-6 and Abeta (seq. 2); TNF and Abeta (seq.3); IL-1b and Abeta (seq.3); Abeta (seq. 1) and Abeta (seq. 3); IGF1,2 and Abeta (seq. 3); Abeta(seq. 3) and IL-18; IL-6 and Abeta (seq. 3); TNF and Abeta (seq. 1);IL-1b and Abeta (seq. 1); IGF1,2 and Abeta (seq. 1); Abeta (seq. 1) andIL-18; IL-6 and Abeta (seq. 1); Abeta (seq. 1) and RAGE; NGF and IL-18;NGF and IL-1b; NGF and IL-6; TNF and EGFR (seq. 2); TNF and RAGE; CD-20and EGFR (seq. 1); CD-20 and EGFR (seq. 2); RGMA and TNF (see Examples2.1 to 2.27)

8. Oncological Disorders

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren, M, et al. (2003) Annu. Rev. Med.54:343-69). Antibodies may exert antitumor effects by inducingapoptosis, redirected cytotoxicity, interfering with ligand-receptorinteractions, or preventing the expression of proteins that are criticalto the neoplastic phenotype. In addition, antibodies can targetcomponents of the tumor microenvironment, perturbing vital structuressuch as the formation of tumor-associated vasculature. Antibodies canalso target receptors whose ligands are growth factors, such as theepidermal growth factor receptor. The antibody thus inhibits naturalligands that stimulate cell growth from binding to targeted tumor cells.Alternatively, antibodies may induce an anti-idiotype network,complement-mediated cytotoxicity, or antibody-dependent cellularcytotoxicity (ADCC). The use of dual-specific antibody that targets twoseparate tumor mediators will likely give additional benefit compared toa mono-specific therapy. In another embodiment, a DVD of the inventionis capable of binding VEGF and phosphatidylserine; VEGF and ErbB3; VEGFand PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP;VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP;IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and CD74; CD20 andCD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGFand c-MET; HGF and NRP1; HGF and phosphatidylserine; ErbB3 and IGF1R;ErbB3 and IGF1,2; c-Met and Her-2; c-Met and NRP1; c-Met and IGF1R;IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2and HER2; IGF2 and CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2;PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met;PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR;RON and c-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 andPLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1;VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and ROBO4;VEGFR2 and CD55; LPA and SIP; EPHB2 and RON; CTLA4 and VEGF; CD3 andEPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA;CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 andCD33; CD80 and CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 andCD4; CD80 and CD52; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22and CD20; CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33;CD22 and CD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF;CD22 and CD52; CD30 and CD20; CD30 and CD22; CD30 and CD23; CD30 andCD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30and DR4; CD30 and VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL;CD33 and FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 andDR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 andDR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 andIGF1,2; DR5 and IGFR, DR5 and HER-2, and EGFR and DLL4. Other targetcombinations include one or more members of the EGF/erb-2/erb-3 family.Other targets (one or more) involved in oncological diseases that DVDIgs may bind include, but are not limited to those selected from thegroup consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A,IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11,FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21,FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2,IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2,FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B,CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1,PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1,EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5,NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3,IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL,KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2,NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2,NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2,FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF,ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18,FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8,FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24,INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5,KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP,SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7,CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2,CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1,AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES,DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1,GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE,PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP,STEAP2, TPM1, TPM2, TRPCδ, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1,FGF2, FIGF, FLT1, JAG1, KDR, LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1,VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMAS, NRP1, NRP2, STAB1,ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1,CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK,EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA,TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV,ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2,CDH1 (E-cadherin), CDKN1B (p27Kipl), CDKN2A (p16INK4a), COL6A1, CTNNB1(b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF),FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST(glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7(c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA),PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A(topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin),CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2(Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19),KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3(metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2),S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2(mammaglobin 1), SPRR1B (Sprl), THBS1, THBS2, THBS4, and TNFAIP2 (B94),RON, c-Met, CD64, DLL4, PLGF, CTLA4, phophatidylserine, ROBO4, CD80,CD22, CD40, CD23, CD28, CD80, CD55, CD38, CD70, CD74, CD30, CD138, CD56,CD33, CD2, CD137, DR4, DR5, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP,EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG,FLT3, PDGFR alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.

IV. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising abinding protein, of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising binding proteins ofthe invention are for use in, but not limited to, diagnosing, detecting,or monitoring a disorder, in preventing, treating, managing, orameliorating of a disorder or one or more symptoms thereof, and/or inresearch. In a specific embodiment, a composition comprises one or morebinding proteins of the invention. In another embodiment, thepharmaceutical composition comprises one or more binding proteins of theinvention and one or more prophylactic or therapeutic agents other thanbinding proteins of the invention for treating a disorder. In anembodiment, the prophylactic or therapeutic agents known to be usefulfor or having been or currently being used in the prevention, treatment,management, or amelioration of a disorder or one or more symptomsthereof. In accordance with these embodiments, the composition mayfurther comprise of a carrier, diluent or excipient.

The binding proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a binding protein ofthe invention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some embodiments, isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, areincluded in the composition. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e. g., Wu and Wu, J. Biol. Chem. 262:4429-4432(1987)), construction of a nucleic acid as part of a retroviral or othervector, etc. Methods of administering a prophylactic or therapeuticagent of the invention include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intraperitoneal,intravenous and subcutaneous), epidurala administration, intratumoraladministration, and mucosal administration (e.g., intranasal and oralroutes). In addition, pulmonary administration can be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309;5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCTPublication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; andWO 99/66903, each of which is incorporated herein by reference theirentireties. In one embodiment, a binding protein of the invention,combination therapy, or a composition of the invention is administeredusing Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.). In a specific embodiment, prophylactic or therapeuticagents of the invention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In an embodiment, specific binding of antibody-coupled carbon nanotubes(CNTs) to tumor cells in vitro, followed by their highly specificablation with near-infrared (NIR) light can be used to target tumorcells. For example, biotinylated polar lipids can be used to preparestable, biocompatible, noncytotoxic CNT dispersions that are thenattached to one or two different neutralite avidin-derivatized DVD-Igsdirected against one or more tumor antigens (e.g., CD22) (Chakravarty,P. et al. (2008) Proc. Natl. Acad. Sci. USA 105:8697-8702.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e. g., one or more prophylactic or therapeutic agents) other than abinding protein of the invention of a subject to prevent, treat, manage,and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253. Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In anembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy &Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science&Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). In an embodiment, for non-sprayable topical dosage forms,viscous to semi-solid or solid forms comprising a carrier or one or moreexcipients compatible with topical application and having a dynamicviscosity greater than water are employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, in an embodiment, in combination with a solid orliquid inert carrier, is packaged in a mixture with a pressurizedvolatile (e.g., a gaseous propellant, such as freon) or in a squeezebottle. Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well-known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO98/31346; and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, a binding proteinof the invention, combination therapy, and/or composition of theinvention is administered using Alkermes AIR® pulmonary drug deliverytechnology (Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e. g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompasse administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. In an embodiment, one or more of the prophylactic ortherapeutic agents or pharmaceutical compositions of the invention issupplied as a dry sterile lyophilized powder in a hermetically sealedcontainer at a unit dosage of at least 5 mg, at least 10 mg, at least 15mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, atleast 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,e.g., within 5 days, within 72 hours, within 48 hours, within 24 hours,within 12 hours, within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,one or more of the prophylactic or therapeutic agents or pharmaceuticalcompositions of the invention is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the agent. In an embodiment, the liquid form of the administeredcomposition is supplied in a hermetically sealed container at least 0.25mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, atleast 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg,at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100mg/ml. The liquid form should be stored at between 2° C. and 8° C. inits original container.

The binding proteins of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration. In anembodiment, the antibody or antibody-portions will be prepared as aninjectable solution containing 0.1-250 mg/ml binding protein. Theinjectable solution can be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampule or pre-filled syringe. Thebuffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0(optimally pH 6.0). Other suitable buffers include but are not limitedto, sodium succinate, sodium citrate, sodium phosphate or potassiumphosphate. Sodium chloride can be used to modify the toxicity of thesolution at a concentration of 0-300 mM (optimally 150 mM for a liquiddosage form). Cryoprotectants can be included for a lyophilized dosageform, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Other suitable bulking agents include glycine, arginine, can be includedas 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additionalsurfactants include but are not limited to polysorbate 20 and BRIJsurfactants. The pharmaceutical composition comprising the bindingproteins of the invention prepared as an injectable solution forparenteral administration, can further comprise an agent useful as anadjuvant, such as those used to increase the absorption, or dispersionof a therapeutic protein (e.g., antibody). A particularly usefuladjuvant is hyaluronidase, such as Hylenex® (recombinant humanhyaluronidase). Addition of hyaluronidase in the injectable solutionimproves human bioavailability following parenteral administration,particularly subcutaneous administration. It also allows for greaterinjection site volumes (i.e. greater than 1 ml) with less pain anddiscomfort, and minimum incidence of injection site reactions. (seeWO2004078140, and US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form chosen depends on the intended mode of administration andtherapeutic application. Typical compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies. Thechosen mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In an embodiment, theantibody is administered by intravenous infusion or injection. Inanother embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated herein, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the methods of preparation are vacuum drying and spray-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The binding proteins of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, in an embodiment, the route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, a binding protein of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding protein of the inventionis coformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders with bindingprotein of the invention. For example, a binding protein of theinvention may be coformulated and/or coadministered with one or moreadditional antibodies that bind other targets (e.g., antibodies thatbind other cytokines or that bind cell surface molecules). Furthermore,one or more antibodies of the invention may be used in combination withtwo or more of the foregoing therapeutic agents. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

In certain embodiments, a binding protein is linked to a half-lifeextending vehicle known in the art. Such vehicles include, but are notlimited to, the Fc domain, polyethylene glycol, and dextran. Suchvehicles are described, e.g., in U.S. application Ser. No. 09/428,082and published PCT Application No. WO 99/25044, which are herebyincorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences encoding a bindingprotein of the invention or another prophylactic or therapeutic agent ofthe invention are administered to treat, prevent, manage, or amelioratea disorder or one or more symptoms thereof by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded antibody orprophylactic or therapeutic agent of the invention that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217;May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY(1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990). Detailed description of various methods ofgene therapy are disclosed in US20050042664 A1 which is incorporatedherein by reference.

The binding proteins of the invention are useful in treating variousdiseases wherein the targets that are recognized by the binding proteinsare detrimental. Such diseases include, but are not limited to,rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia areata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-l-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aordic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chromic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti.system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occulsive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue. (see Peritt et al. PCTpublication No. WO2002097048A2, Leonard et al., PCT publication No.WO9524918 A1, and Salfeld et al., PCT publication No. WO00/56772A1).

The DVD-Igs of the invention may also treat one or more of the followingdiseases: Acute coronary syndromes, Acute Idiopathic Polyneuritis, AcuteInflammatory Demyelinating Polyradiculoneuropathy, Acute ischemia, AdultStill's Disease, Alopecia areata, Anaphylaxis, Anti-PhospholipidAntibody Syndrome, Aplastic anemia, Arteriosclerosis, Atopic eczema,Atopic dermatitis, Autoimmune dermatitis, Autoimmune disorder associatedwith Streptococcus infection, Autoimmune hearingloss, AutoimmuneLymphoproliferative Syndrome (ALPS), Autoimmune myocarditis, autoimmunethrombocytopenia (AITP), Blepharitis, Bronchiectasis, Bullouspemphigoid, Cardiovascular Disease, Catastrophic AntiphospholipidSyndrome, Celiac Disease, Cervical Spondylosis, Chronic ischemia,Cicatricial pemphigoid, Clinically isolated Syndrome (CIS) with Risk forMultiple Sclerosis, Conjunctivitis, Childhood Onset PsychiatricDisorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis,dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Diskherniation, Disk prolaps, Drug induced immune hemolytic anemia,Endocarditis, Endometriosis, endophthalmitis, Episcleritis, Erythemamultiforme, erythema multiforme major, Gestational pemphigoid,Guillain-Barré Syndrome (GBS), Hay Fever, Hughes Syndrome, IdiopathicParkinson's Disease, idiopathic interstitial pneumonia, IgE-mediatedAllergy, Immune hemolytic anemia, Inclusion Body Myositis, Infectiousocular inflammatory disease, Inflammatory demyelinating disease,Inflammatory heart disease, Inflammatory kidney disease, IPF/UIP,Iritis, Keratitis, Keratojuntivitis sicca, Kussmaul disease orKussmaul-Meier Disease, Landry's Paralysis, Langerhan's CellHistiocytosis, Livedo reticularis, Macular Degeneration, malignancies,Microscopic Polyangiitis, Morbus Bechterev, Motor Neuron Disorders,Mucous membrane pemphigoid, Multiple Organ failure, Myasthenia Gravis,Myelodysplastic Syndrome, Myocarditis, Nerve Root Disorders, Neuropathy,Non-A Non-B Hepatitis, Optic Neuritis, Osteolysis, Ovarian cancer,Pauciarticular JRA, peripheral artery occlusive disease (PAOD),peripheral vascular disease (PVD), peripheral artery disease (PAD),Phlebitis, Polyarteritis nodosa (or periarteritis nodosa),Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA,Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica(PMR), Post-Pump Syndrome, primary parkinsonism, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency,Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease,SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis),Scleroderma, Secondary Amyloidosis, Shock lung, Scleritis, Sciatica,Secondary Adrenal Insufficiency, Silicone associated connective tissuedisease, Sneddon-Wilkinson Dermatosis, spondilitis ankylosans,Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome,Temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1allergic reaction, Type II Diabetes, Urticaria, Usual interstitialpneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular degeneration,and Wound healing.

The binding proteins of the invention can be used to treat humanssuffering from autoimmune diseases, in particular those associated withinflammation, including, rheumatoid arthritis, spondylitis, allergy,autoimmune diabetes, autoimmune uveitis. In an embodiment, the bindingproteins of the invention or antigen-binding portions thereof, are usedto treat rheumatoid arthritis, Crohn's disease, multiple sclerosis,insulin dependent diabetes mellitus and psoriasis.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods of the invention include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In an embodiment, the antibodies of the invention or antigen-bindingportions thereof, are used to treat cancer or in the prevention ofmetastases from the tumors described herein either when used alone or incombination with radiotherapy and/or other chemotherapeutic agents.

The antibodies of the invention, or antigen binding portions thereof,may be combined with agents that include but are not limited to,antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylatingagents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines,adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors,5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinaseinhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g.,celecoxib), kinase inhibitors, and siRNAs.

A binding protein of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of variousdiseases.

A binding protein of the invention can be used alone or in combinationto treat such diseases. It should be understood that the bindingproteins can be used alone or in combination with an additional agent,e.g., a therapeutic agent, said additional agent being selected by theskilled artisan for its intended purpose. For example, the additionalagent can be a therapeutic agent art-recognized as being useful to treatthe disease or condition being treated by the antibody of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition e.g., an agent whicheffects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Combinations to treat autoimmune and inflammatory diseases arenon-steroidal anti-inflammatory drug(s) also referred to as NSAIDS whichinclude drugs like ibuprofen. Other combinations are corticosteroidsincluding prednisolone; the well known side-effects of steroid use canbe reduced or even eliminated by tapering the steroid dose required whentreating patients in combination with the DVD Igs of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons,EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Combinations of therapeutic agents may interfere at different points inthe autoimmune and subsequent inflammatory cascade; examples include TNFantagonists like chimeric, humanized or human TNF antibodies,Adalimumab, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571,and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG(Enbrel™) or p55TNFR1gG (Lenercept), and also TNFα converting enzyme(TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-convertingenzyme inhibitors, IL-1RA etc.) may be effective for the same reason.Other combinations include Interleukin 11. Yet another combinationinclude key players of the autoimmune response which may act parallelto, dependent on or in concert with IL-12 function; especially are IL-18antagonists including IL-18 antibodies or soluble IL-18 receptors, orIL-18 binding proteins. It has been shown that IL-12 and IL-18 haveoverlapping but distinct functions and a combination of antagonists toboth may be most effective. Yet another combination are non-depletinganti-CD4 inhibitors. Yet other combinations include antagonists of theco-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies,soluble receptors or antagonistic ligands.

The binding proteins of the invention may also be combined with agents,such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine,olsalazine chloroquinine/hydroxychloroquine, pencillamine,aurothiomalate (intramuscular and oral), azathioprine, cochicine,corticosteroids (oral, inhaled and local injection), beta-2adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroidssuch as prednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors),IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE)inhibitors, T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid,hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen,valdecoxib, sulfasalazine, methylprednisolone, meloxicam,methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenacsodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl,salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen,alendronate sodium, prednisolone, morphine sulfate, lidocainehydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptylinehcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab,IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796,SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, andMesopram. Combinations include methotrexate or leflunomide and inmoderate or severe rheumatoid arthritis cases, cyclosporine.

Nonlimiting additional agents which can also be used in combination witha binding protein to treat rheumatoid arthritis include, but are notlimited to, the following: non-steroidal anti-inflammatory drug(s)(NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs);CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer);cA2/infliximab (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex;see e.g., Arthritis & Rheumatism (1994) Vol. 37, 5295; J. Invest. Med.(1996) Vol. 44, 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusionprotein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depletingprimatized anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis &Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2fusion proteins; Seragen; see e.g., Arthritis & Rheumatism (1993) Vol.36, 1223); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche);IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen);TNF-bp/s-TNF (soluble TNF binding protein; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), 5284; Amer. J.Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42);R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996)Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 5282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), 5131; Inflammation Research (1996) Vol. 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284);T-614 (cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp.1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor ofthe enzyme interleukin-1β converting enzyme); zap-70 and/or lckinhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGFinhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptor;inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies;anti-IL-18 antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S308);interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S120); gold; penicillamine; chloroquine;chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; totallymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies;CD5-toxins; orally-administered peptides and collagen; lobenzaritdisodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioateoligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone;orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2Rantibodies; marine and botanical lipids (fish and plant seed fattyacids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am.21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamicacid; intravenous immune globulin; zileuton; azaribine; mycophenolicacid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2inhibitors (see Bruncko, Milan et al., Journal of Medicinal Chemistry(2007), 50(4), 641-662); antivirals and immune modulating agents.

In one embodiment, the binding protein or antigen-binding portionthereof, is administered in combination with one of the following agentsfor the treatment of rheumatoid arthritis: small molecule inhibitor ofKDR, small molecule inhibitor of Tie-2; methotrexate; prednisone;celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib;etanercept; infliximab; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hcl; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15;BIRB-796; SC10-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; and mesopram.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which a binding protein of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs;growth factors; elastase inhibitors; pyridinyl-imidazole compounds;antibodies to or antagonists of other human cytokines or growth factors,for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17,IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD90 or their ligands. The antibodies of theinvention, or antigen binding portions thereof, may also be combinedwith agents, such as methotrexate, cyclosporin, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFαconverting enzyme inhibitors, T-cell signalling inhibitors such askinase inhibitors, metalloproteinase inhibitors, sulfasalazine,azathioprine, 6-mercaptopurines, angiotensin converting enzymeinhibitors, soluble cytokine receptors and derivatives thereof (e.g.,soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) andantiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ)and bcl-2 inhibitors.

Examples of therapeutic agents for Crohn's disease in which a bindingprotein can be combined include the following: TNF antagonists, forexample, anti-TNF antibodies, Adalimumab (PCT Publication No. WO97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs,(p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) inhibitors and PDE4inhibitors. Antibodies of the invention, or antigen binding portionsthereof, can be combined with corticosteroids, for example, budenosideand dexamethasone. Binding proteins of the invention or antigen bindingportions thereof, may also be combined with agents such assulfasalazine, 5-aminosalicylic acid and olsalazine, and agents whichinterfere with synthesis or action of proinflammatory cytokines such asIL-1, for example, IL-1β converting enzyme inhibitors and IL-1ra.Antibodies of the invention or antigen binding portion thereof may alsobe used with T cell signaling inhibitors, for example, tyrosine kinaseinhibitors 6-mercaptopurines. Binding proteins of the invention, orantigen binding portions thereof, can be combined with IL-11. Bindingproteins of the invention, or antigen binding portions thereof, can becombined with mesalamine, prednisone, azathioprine, mercaptopurine,infliximab, methylprednisolone sodium succinate, diphenoxylate/atropsulfate, loperamide hydrochloride, methotrexate, omeprazole, folate,ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracyclinehydrochloride, fluocinonide, metronidazole, thimerosal/boric acid,cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyaminesulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodonehcl/acetaminophen, promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab and interferon-gamma

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich binding proteins of the invention can be combined include thefollowing: corticosteroids; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; methotrexate;4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen);interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (InterferonSciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferonβ1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; clabribine; antibodies to or antagonists of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, and PDGF. Binding proteins of the invention can be combinedwith antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. Binding proteins of the invention, may also be combinedwith agents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adensosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g.,IL-4, IL-10, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents for multiple sclerosis in which bindingproteins of the invention can be combined include interferon-β, forexample, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspaseinhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNFinhibitors, and antibodies to CD40 ligand and CD80.

The binding proteins of the invention, may also be combined with agents,such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab,sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokinereceptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposomeencapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298,mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody,neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel,teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example,TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gammaantagonists, IL-4 agonists.

Non-limiting examples of therapeutic agents for Angina with whichbinding proteins of the invention can be combined include the following:aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate,atenolol, metoprolol tartrate, amlodipine besylate, diltiazemhydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine,atorvastatin calcium, potassium chloride, furosemide, simvastatin,verapamil hcl, digoxin, propranolol hydrochloride, carvedilol,lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate,nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprololfumarate.

Non-limiting examples of therapeutic agents for Ankylosing Spondylitiswith which binding proteins of the invention can be combined include thefollowing: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam,indomethacin, diclofenac, celecoxib, rofecoxib, Sulfasalazine,Methotrexate, azathioprine, minocyclin, prednisone, etanercept,infliximab.

Non-limiting examples of therapeutic agents for Asthma with whichbinding proteins of the invention can be combined include the following:albuterol, salmeterol/fluticasone, montelukast sodium, fluticasonepropionate, budesonide, prednisone, salmeterol xinafoate, levalbuterolhcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate,triamcinolone acetonide, beclomethasone dipropionate, ipratropiumbromide, azithromycin, pirbuterol acetate, prednisolone, theophyllineanhydrous, methylprednisolone sodium succinate, clarithromycin,zafirlukast, formoterol fumarate, influenza virus vaccine,methylprednisolone, amoxicillin trihydrate, flunisolide, allergyinjection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which bindingproteins of the invention can be combined include the following:albuterol sulfate/ipratropium, ipratropium bromide,salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasonepropionate, prednisone, theophylline anhydrous, methylprednisolonesodium succinate, montelukast sodium, budesonide, formoterol fumarate,triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxonesodium, amoxicillin trihydrate, gatifloxacin, zafirlukast,amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents for HCV with which bindingproteins of the invention can be combined include the following:Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1,Interferon-alpha-n1, Pegylated interferon-alpha-2a, Pegylatedinterferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin,Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497and any compounds that are used to treat HCV through intervention withthe following targets: HCV polymerase, HCV protease, HCV helicase, HCVIRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for Idiopathic PulmonaryFibrosis with which binding proteins of the invention can be combinedinclude the following: prednisone, azathioprine, albuterol, colchicine,albuterol sulfate, digoxin, gamma interferon, methylprednisolone sodsucc, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone,cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,fluticasone propionate, levofloxacin, metaproterenol sulfate, morphinesulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide,tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,mycophenolate mofetil, Interferon-gamma-1β.

Non-limiting examples of therapeutic agents for Myocardial Infarctionwith which binding proteins of the invention can be combined include thefollowing: aspirin, nitroglycerin, metoprolol tartrate, enoxaparinsodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol,morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril,isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,tenecteplase, enalapril maleate, torsemide, retavase, losartanpotassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat,amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazemhydrochloride, captopril, irbesartan, valsartan, propranololhydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone,interferon, sotalol hydrochloride, potassium chloride, docusate sodium,dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium,midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, cariporide.

Non-limiting examples of therapeutic agents for Psoriasis with whichbinding proteins of the invention can be combined include the following:small molecule inhibitor of KDR, small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, pimecrolimus, coal tar, diflorasone diacetate, etanerceptfolate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,methylprednisolone acetate, prednisone, sunscreen, halcinonide,salicylic acid, anthralin, clocortolone pivalate, coal extract, coaltar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone,diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/nalact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen,salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,pimecrolimus, PUVA, UVB, sulfasalazine.

Non-limiting examples of therapeutic agents for Psoriatic Arthritis withwhich binding proteins of the invention can be combined include thefollowing: methotrexate, etanercept, rofecoxib, celecoxib, folic acid,sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,betamethasone diprop augmented, infliximab, methotrexate, folate,triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenacsodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodiumthiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium,sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2inhibitors.

Non-limiting examples of therapeutic agents for Restenosis with whichbinding proteins of the invention can be combined include the following:sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus,acetaminophen.

Non-limiting examples of therapeutic agents for Sciatica with whichbinding proteins of the invention can be combined include the following:hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl,methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen,celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeinephosphate/apap, tramadol hcl/acetaminophen, metaxalone, meloxicam,methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin,dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,diclofenac sodium/misoprostol, propoxyphene napsylate/apap,asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeinephos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrinecitrate, temazepam.

Examples of therapeutic agents for SLE (Lupus) in which binding proteinsof the invention can be combined include the following: NSAIDS, forexample, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2inhibitors, for example, Celecoxib, rofecoxib, valdecoxib;anti-malarials, for example, hydroxychloroquine; Steroids, for example,prednisone, prednisolone, budenoside, dexamethasone; Cytotoxics, forexample, azathioprine, cyclophosphamide, mycophenolate mofetil,methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, forexample Cellcept. Binding proteins of the invention, may also becombined with agents such as sulfasalazine, 5-aminosalicylic acid,olsalazine, Imuran and agents which interfere with synthesis, productionor action of proinflammatory cytokines such as IL-1, for example,caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra.Binding proteins of the invention may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors; or molecules thattarget T cell activation molecules, for example, CTLA-4-IgG or anti-B7family antibodies, anti-PD-1 family antibodies. Binding proteins of theinvention, can be combined with IL-11 or anti-cytokine antibodies, forexample, fonotolizumab (anti-IFNg antibody), or anti-receptor receptorantibodies, for example, anti-IL-6 receptor antibody and antibodies toB-cell surface molecules. Antibodies of the invention or antigen bindingportion thereof may also be used with LIP 394 (abetimus), agents thatdeplete or inactivate B-cells, for example, Rituximab (anti-CD20antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, forexample, anti-TNF antibodies, Adalimumab (PCT Publication No. WO97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs,(p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) and bcl-2 inhibitors,because bcl-2 overexpression in transgenic mice has been demonstrated tocause a lupus like phenotype (see Marquina, Regina et al., Journal ofImmunology (2004), 172(11), 7177-7185), therefore inhibition is expectedto have therapeutic effects.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody, orantibody portion, are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a binding protein of the inventionis 0.1-20 mg/kg, for example, 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

V. Diagnostics

The disclosure herein also provides diagnostic applications. This isfurther elucidated below.

I. Method of Assay

The present disclosure also provides a method for determining thepresence, amount or concentration of an analyte (or a fragment thereof)in a test sample using at least one DVD-Ig as described herein. Anysuitable assay as is known in the art can be used in the method.Examples include, but are not limited to, immunoassay, such as sandwichimmunoassay (e.g., monoclonal, polyclonal and/or DVD-Ig sandwichimmunoassays or any variation thereof (e.g., monoclonal/DVD-Ig,DVD-Ig/polyclonal, etc.), including radioisotope detection(radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA)or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISAassays, R&D Systems, Minneapolis, Minn.))), competitive inhibitionimmunoassay (e.g., forward and reverse), fluorescence polarizationimmunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT),bioluminescence resonance energy transfer (BRET), and homogeneouschemiluminescent assay, etc. In a SELDI-based immunoassay, a capturereagent that specifically binds an analyte (or a fragment thereof) ofinterest is attached to the surface of a mass spectrometry probe, suchas a pre-activated protein chip array. The analyte (or a fragmentthereof) is then specifically captured on the biochip, and the capturedanalyte (or a fragment thereof) is detected by mass spectrometry.Alternatively, the analyte (or a fragment thereof) can be eluted fromthe capture reagent and detected by traditional MALDI (matrix-assistedlaser desorption/ionization) or by SELDI. A chemiluminescentmicroparticle immunoassay, in particular one employing the ARCHITECT®automated analyzer (Abbott Laboratories, Abbott Park, IL), is an exampleof a preferred immunoassay.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when a DVD-Ig asdescribed herein is employed as an immunodiagnostic reagent and/or in ananalyte immunoassay kit. The test sample can comprise further moietiesin addition to the analyte of interest, such as antibodies, antigens,haptens, hormones, drugs, enzymes, receptors, proteins, peptides,polypeptides, oligonucleotides and/or polynucleotides. For example, thesample can be a whole blood sample obtained from a subject. It can benecessary or desired that a test sample, particularly whole blood, betreated prior to immunoassay as described herein, e.g., with apretreatment reagent. Even in cases where pretreatment is not necessary(e.g., most urine samples), pretreatment optionally can be done (e.g.,as part of a regimen on a commercial platform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassay and kits of the invention. The pretreatment optionallycomprises: (a) one or more solvents (e.g., methanol and ethylene glycol)and optionally, salt, (b) one or more solvents and salt, and optionally,detergent, (c) detergent, or (d) detergent and salt. Pretreatmentreagents are known in the art, and such pretreatment can be employed,e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers(Abbott Laboratories, Abbott Park, IL), as described in the literature(see, e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody AssayEvaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36:1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYMCyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMITCyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or ascommercially available. Additionally, pretreatment can be done asdescribed in Abbott's U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0471 293, U.S. Provisional Pat. App. 60/878,017, filed Dec. 29, 2006, andU.S. Pat. App. Pub. No. 2008/0020401 (incorporated by reference in itsentirety for its teachings regarding pretreatment). The pretreatmentreagent can be a heterogeneous agent or a homogeneous agent.

With use of a heterogeneous pretreatment reagent, the pretreatmentreagent precipitates analyte binding protein (e.g., protein that canbind to an analyte or a fragment thereof) present in the sample. Such apretreatment step comprises removing any analyte binding protein byseparating from the precipitated analyte binding protein the supernatantof the mixture formed by addition of the pretreatment agent to sample.In such an assay, the supernatant of the mixture absent any bindingprotein is used in the assay, proceeding directly to the antibodycapture step.

With use of a homogeneous pretreatment reagent there is no suchseparation step. The entire mixture of test sample and pretreatmentreagent are contacted with a labeled specific binding partner foranalyte (or a fragment thereof), such as a labeled anti-analyte antibody(or an antigenically reactive fragment thereof). The pretreatmentreagent employed for such an assay typically is diluted in thepretreated test sample mixture, either before or during capture by thefirst specific binding partner. Despite such dilution, a certain amountof the pretreatment reagent is still present (or remains) in the testsample mixture during capture. According to the invention, the labeledspecific binding partner can be a DVD-Ig (or a fragment, a variant, or afragment of a variant thereof).

In a heterogeneous format, after the test sample is obtained from asubject, a first mixture is prepared. The mixture contains the testsample being assessed for an analyte (or a fragment thereof) and a firstspecific binding partner, wherein the first specific binding partner andany analyte contained in the test sample form a first specific bindingpartner-analyte complex.

Preferably, the first specific binding partner is an anti-analyteantibody or a fragment thereof. The first specific binding partner canbe a DVD-Ig (or a fragment, a variant, or a fragment of a variantthereof) as described herein. The order in which the test sample and thefirst specific binding partner are added to form the mixture is notcritical. Preferably, the first specific binding partner is immobilizedon a solid phase. The solid phase used in the immunoassay (for the firstspecific binding partner and, optionally, the second specific bindingpartner) can be any solid phase known in the art, such as, but notlimited to, a magnetic particle, a bead, a test tube, a microtiterplate, a cuvette, a membrane, a scaffolding molecule, a film, a filterpaper, a disc and a chip.

After the mixture containing the first specific binding partner-analytecomplex is formed, any unbound analyte is removed from the complex usingany technique known in the art. For example, the unbound analyte can beremoved by washing. Desirably, however, the first specific bindingpartner is present in excess of any analyte present in the test sample,such that all analyte that is present in the test sample is bound by thefirst specific binding partner.

After any unbound analyte is removed, a second specific binding partneris added to the mixture to form a first specific bindingpartner-analyte-second specific binding partner complex. The secondspecific binding partner is preferably an anti-analyte antibody thatbinds to an epitope on analyte that differs from the epitope on analytebound by the first specific binding partner. Moreover, also preferably,the second specific binding partner is labeled with or contains adetectable label as described above. The second specific binding partnercan be a DVD-Ig (or a fragment, a variant, or a fragment of a variantthereof) as described herein.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as 3H,125I, 35S, 14C, 32P, and 33P), an enzymatic label (such as horseradishperoxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, andthe like), a chemiluminescent label (such as acridinium esters,thioesters, or sulfonamides; luminol, isoluminol, phenanthridiniumesters, and the like), a fluorescent label (such as fluorescein (e.g.,5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)),rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zincsulfide-capped cadmium selenide), a thermometric label, or animmuno-polymerase chain reaction label. An introduction to labels,labeling procedures and detection of labels is found in Polak and VanNoorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag,N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes andResearch Chemicals (1996), which is a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. A fluorescent labelcan be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904,5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated byreference in their entireties). An acridinium compound can be used as adetectable label in a homogeneous or heterogeneous chemiluminescentassay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Left. 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317(2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004);and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).

A preferred acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem.63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914(1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In LuminescenceBiotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press:Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782(2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each ofwhich is incorporated herein by reference in its entirety for itsteachings regarding same). Another preferred acridinium compound is anacridinium-9-carboxylate aryl ester. An example of anacridinium-9-carboxylate aryl ester is10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available fromCayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium9-carboxylate aryl esters are described in McCapra et al., Photochem.Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249(2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Pat.No. 5,241,070 (each of which is incorporated herein by reference in itsentirety for its teachings regarding same). Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in US2008-0248493.

Chemiluminescent assays (e.g., using acridinium as described above orother chemiluminescent agents) can be performed in accordance with themethods described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67(2006). While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith a chemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte complexes form.Alternatively, if a second specific binding partner is used and thesecond specific binding partner is detectably labeled with achemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte-second specific bindingpartner complexes form. Any unbound specific binding partner, whetherlabeled or unlabeled, can be removed from the mixture using anytechnique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture (e.g., the source of the hydrogen peroxide beingone or more buffers or other solutions that are known to containhydrogen peroxide) before, simultaneously with, or after the addition ofan above-described acridinium compound. Hydrogen peroxide can begenerated in situ in a number of ways such as would be apparent to oneskilled in the art.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of analyte in the samplecan be quantified. Specifically, the amount of analyte in the sample isproportional to the intensity of the signal generated. The amount ofanalyte present can be quantified by comparing the amount of lightgenerated to a standard curve for analyte or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions of known concentrations of analyte by massspectroscopy, gravimetric methods, and other techniques known in theart. While the above is described with emphasis on use of an acridiniumcompound as the chemiluminescent agent, one of ordinary skill in the artcan readily adapt this description for use of other chemiluminescentagents.

Analyte immunoassays generally can be conducted using any format knownin the art, such as, but not limited to, a sandwich format.Specifically, in one immunoassay format, at least two antibodies areemployed to separate and quantify analyte, such as human analyte, or afragment thereof in a sample. More specifically, the at least twoantibodies bind to different epitopes on an analyte (or a fragmentthereof) forming an immune complex, which is referred to as a“sandwich.” Generally, in the immunoassays one or more antibodies can beused to capture the analyte (or a fragment thereof) in the test sample(these antibodies are frequently referred to as a “capture” antibody or“capture” antibodies) and one or more antibodies can be used to bind adetectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection antibody,” the“detection antibodies,” the “conjugate,” or the “conjugates”). Thus, inthe context of a sandwich immunoassay format, a DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof) as described herein can beused as a capture antibody, a detection antibody, or both. For example,one DVD-Ig having a domain that can bind a first epitope on an analyte(or a fragment thereof) can be used as a capture antibody and/or anotherDVD-Ig having a domain that can bind a second epitope on an analyte (ora fragment thereof) can be used as a detection antibody. In this regard,a DVD-Ig having a first domain that can bind a first epitope on ananalyte (or a fragment thereof) and a second domain that can bind asecond epitope on an analyte (or a fragment thereof) can be used as acapture antibody and/or a detection antibody. Alternatively, one DVD-Ighaving a first domain that can bind an epitope on a first analyte (or afragment thereof) and a second domain that can bind an epitope on asecond analyte (or a fragment thereof) can be used as a capture antibodyand/or a detection antibody to detect, and optionally quantify, two ormore analytes. In the event that an analyte can be present in a samplein more than one form, such as a monomeric form and a dimeric/multimericform, which can be homomeric or heteromeric, one DVD-Ig having a domainthat can bind an epitope that is only exposed on the monomeric form andanother DVD-Ig having a domain that can bind an epitope on a differentpart of a dimeric/multimeric form can be used as capture antibodiesand/or detection antibodies, thereby enabling the detection, andoptional quantification, of different forms of a given analyte.Furthermore, employing DVD-Igs with differential affinities within asingle DVD-Ig and/or between DVD-Igs can provide an avidity advantage.In the context of immunoassays as described herein, it generally may behelpful or desired to incorporate one or more linkers within thestructure of a DVD-Ig. When present, optimally the linker should be ofsufficient length and structural flexibility to enable binding of anepitope by the inner domains as well as binding of another epitope bythe outer domains. In this regard, if a DVD-Ig can bind two differentanalytes and one analyte is larger than the other, desirably the largeranalyte is bound by the outer domains.

Generally speaking, a sample being tested for (for example, suspected ofcontaining) analyte (or a fragment thereof) can be contacted with atleast one capture antibody (or antibodies) and at least one detectionantibody (which can be a second detection antibody or a third detectionantibody or even a successively numbered antibody, e.g., as where thecapture and/or detection antibody comprise multiple antibodies) eithersimultaneously or sequentially and in any order. For example, the testsample can be first contacted with at least one capture antibody andthen (sequentially) with at least one detection antibody. Alternatively,the test sample can be first contacted with at least one detectionantibody and then (sequentially) with at least one capture antibody. Inyet another alternative, the test sample can be contacted simultaneouslywith a capture antibody and a detection antibody.

In the sandwich assay format, a sample suspected of containing analyte(or a fragment thereof) is first brought into contact with at least onefirst capture antibody under conditions that allow the formation of afirst antibody/analyte complex. If more than one capture antibody isused, a first capture antibody/analyte complex comprising two or morecapture antibodies is formed. In a sandwich assay, the antibodies, i.e.,preferably, the at least one capture antibody, are used in molar excessamounts of the maximum amount of analyte (or a fragment thereof)expected in the test sample. For example, from about 5 μg to about 1 mgof antibody per mL of buffer (e.g., microparticle coating buffer) can beused.

Competitive inhibition immunoassays, which are often used to measuresmall analytes because binding by only one antibody is required,comprise sequential and classic formats. In a sequential competitiveinhibition immunoassay a capture antibody to an analyte of interest iscoated onto a well of a microtiter plate or other solid support. Whenthe sample containing the analyte of interest is added to the well, theanalyte of interest binds to the capture antibody. After washing, aknown amount of labeled (e.g., biotin or horseradish peroxidase (HRP))analyte is added to the well. A substrate for an enzymatic label isnecessary to generate a signal. An example of a suitable substrate forHRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signalgenerated by the labeled analyte is measured and is inverselyproportional to the amount of analyte in the sample. In a classiccompetitive inhibition immunoassay an antibody to an analyte of interestis coated onto a solid support (e.g., a well of a microtiter plate).However, unlike the sequential competitive inhibition immunoassay, thesample and the labeled analyte are added to the well at the same time.Any analyte in the sample competes with labeled analyte for binding tothe capture antibody. After washing, the signal generated by the labeledanalyte is measured and is inversely proportional to the amount ofanalyte in the sample.

Optionally, prior to contacting the test sample with the at least onecapture antibody (for example, the first capture antibody), the at leastone capture antibody can be bound to a solid support, which facilitatesthe separation of the first antibody/analyte (or a fragment thereof)complex from the test sample. The substrate to which the captureantibody is bound can be any suitable solid support or solid phase thatfacilitates separation of the capture antibody-analyte complex from thesample.

Examples include a well of a plate, such as a microtiter plate, a testtube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin), apolymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads ormagnetic beads), a strip of a filter/membrane (e.g., nitrocellulose ornylon), microparticles (e.g., latex particles, magnetizablemicroparticles (e.g., microparticles having ferric oxide or chromiumoxide cores and homo- or hetero-polymeric coats and radii of about 1-10microns). The substrate can comprise a suitable porous material with asuitable surface affinity to bind antigens and sufficient porosity toallow access by detection antibodies. A microporous material isgenerally preferred, although a gelatinous material in a hydrated statecan be used. Such porous substrates are preferably in the form of sheetshaving a thickness of about 0.01 to about 0.5 mm, preferably about 0.1mm. While the pore size may vary quite a bit, preferably the pore sizeis from about 0.025 to about 15 microns, more preferably from about 0.15to about 15 microns. The surface of such substrates can be activated bychemical processes that cause covalent linkage of an antibody to thesubstrate. Irreversible binding, generally by adsorption throughhydrophobic forces, of the antigen or the antibody to the substrateresults; alternatively, a chemical coupling agent or other means can beused to bind covalently the antibody to the substrate, provided thatsuch binding does not interfere with the ability of the antibody to bindto analyte. Alternatively, the antibody can be bound withmicroparticles, which have been previously coated with streptavidin(e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad, Calif.) or biotin(e.g., using Power-Bind™-SA-MP streptavidin-coated microparticles(Seradyn, Indianapolis, Ind.)) or anti-species-specific monoclonalantibodies. If necessary, the substrate can be derivatized to allowreactivity with various functional groups on the antibody. Suchderivatization requires the use of certain coupling agents, examples ofwhich include, but are not limited to, maleic anhydride,N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or more capture reagents, such asantibodies (or fragments thereof), each of which is specific foranalyte(s) can be attached to solid phases in different physical oraddressable locations (e.g., such as in a biochip configuration (see,e.g., U.S. Pat. No. 6,225,047; Int'l Pat. App. Pub. No. WO 99/51773;U.S. Pat. No. 6,329,209; Int'l Pat. App. Pub. No. WO 00/56934, and U.S.Pat. No. 5,242,828). If the capture reagent is attached to a massspectrometry probe as the solid support, the amount of analyte bound tothe probe can be detected by laser desorption ionization massspectrometry. Alternatively, a single column can be packed withdifferent beads, which are derivatized with the one or more capturereagents, thereby capturing the analyte in a single place (see,antibody-derivatized, bead-based technologies, e.g., the xMAP technologyof Luminex (Austin, Tex.)).

After the test sample being assayed for analyte (or a fragment thereof)is brought into contact with the at least one capture antibody (forexample, the first capture antibody), the mixture is incubated in orderto allow for the formation of a first antibody (or multipleantibody)-analyte (or a fragment thereof) complex. The incubation can becarried out at a pH of from about 4.5 to about 10.0, at a temperature offrom about 2° C. to about 45° C., and for a period from at least aboutone (1) minute to about eighteen (18) hours, preferably from about 1 toabout 24 minutes, most preferably for about 4 to about 18 minutes. Theimmunoassay described herein can be conducted in one step (meaning thetest sample, at least one capture antibody and at least one detectionantibody are all added sequentially or simultaneously to a reactionvessel) or in more than one step, such as two steps, three steps, etc.

After formation of the (first or multiple) capture antibody/analyte (ora fragment thereof) complex, the complex is then contacted with at leastone detection antibody under conditions which allow for the formation ofa (first or multiple) capture antibody/analyte (or a fragmentthereof)/second detection antibody complex). While captioned for clarityas the “second” antibody (e.g., second detection antibody), in fact,where multiple antibodies are used for capture and/or detection, the atleast one detection antibody can be the second, third, fourth, etc.antibodies used in the immunoassay. If the capture antibody/analyte (ora fragment thereof) complex is contacted with more than one detectionantibody, then a (first or multiple) capture antibody/analyte (or afragment thereof)/(multiple) detection antibody complex is formed. Aswith the capture antibody (e.g., the first capture antibody), when theat least one (e.g., second and any subsequent) detection antibody isbrought into contact with the capture antibody/analyte (or a fragmentthereof) complex, a period of incubation under conditions similar tothose described above is required for the formation of the (first ormultiple) capture antibody/analyte (or a fragment thereof)/(second ormultiple) detection antibody complex. Preferably, at least one detectionantibody contains a detectable label. The detectable label can be boundto the at least one detection antibody (e.g., the second detectionantibody) prior to, simultaneously with, or after the formation of the(first or multiple) capture antibody/analyte (or a fragmentthereof)/(second or multiple) detection antibody complex. Any detectablelabel known in the art can be used (see discussion above, including ofthe Polak and Van Noorden (1997) and Haugland (1996) references).

The detectable label can be bound to the antibodies either directly orthrough a coupling agent. An example of a coupling agent that can beused is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,hydrochloride), which is commercially available from Sigma-Aldrich, St.Louis, Mo. Other coupling agents that can be used are known in the art.Methods for binding a detectable label to an antibody are known in theart. Additionally, many detectable labels can be purchased orsynthesized that already contain end groups that facilitate the couplingof the detectable label to the antibody, such as CPSP-Acridinium Ester(i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) or SPSP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The (first or multiple) capture antibody/analyte/(second or multiple)detection antibody complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the at least one capture antibody (e.g., thefirst capture antibody) is bound to a solid support, such as a well or abead, separation can be accomplished by removing the fluid (of the testsample) from contact with the solid support. Alternatively, if the atleast first capture antibody is bound to a solid support, it can besimultaneously contacted with the analyte-containing sample and the atleast one second detection antibody to form a first (multiple)antibody/analyte/second (multiple) antibody complex, followed by removalof the fluid (test sample) from contact with the solid support. If theat least one first capture antibody is not bound to a solid support,then the (first or multiple) capture antibody/analyte/(second ormultiple) detection antibody complex does not have to be removed fromthe test sample for quantification of the amount of the label.

After formation of the labeled capture antibody/analyte/detectionantibody complex (e.g., the first capture antibody/analyte/seconddetection antibody complex), the amount of label in the complex isquantified using techniques known in the art. For example, if anenzymatic label is used, the labeled complex is reacted with a substratefor the label that gives a quantifiable reaction such as the developmentof color. If the label is a radioactive label, the label is quantifiedusing appropriate means, such as a scintillation counter. If the labelis a fluorescent label, the label is quantified by stimulating the labelwith a light of one color (which is known as the “excitationwavelength”) and detecting another color (which is known as the“emission wavelength”) that is emitted by the label in response to thestimulation. If the label is a chemiluminescent label, the label isquantified by detecting the light emitted either visually or by usingluminometers, x-ray film, high speed photographic film, a CCD camera,etc. Once the amount of the label in the complex has been quantified,the concentration of analyte or a fragment thereof in the test sample isdetermined by appropriate means, such as by use of a standard curve thathas been generated using serial dilutions of analyte or a fragmentthereof of known concentration. Other than using serial dilutions ofanalyte or a fragment thereof, the standard curve can be generatedgravimetrically, by mass spectroscopy and by other techniques known inthe art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should be about 6.0+/−0.2, themicroparticle coating buffer should be maintained at about roomtemperature (i.e., at from about 17 to about 27° C.), the microparticlecoating buffer pH should be about 6.5+/−0.2, and the microparticlediluent pH should be about 7.8+/−0.2. Solids preferably are less thanabout 0.2%, such as less than about 0.15%, less than about 0.14%, lessthan about 0.13%, less than about 0.12%, or less than about 0.11%, suchas about 0.10%.

FPIAs are based on competitive binding immunoassay principles. Afluorescently labeled compound, when excited by a linearly polarizedlight, will emit fluorescence having a degree of polarization inverselyproportional to its rate of rotation. When a fluorescently labeledtracer-antibody complex is excited by a linearly polarized light, theemitted light remains highly polarized because the fluorophore isconstrained from rotating between the time light is absorbed and thetime light is emitted. When a “free” tracer compound (i.e., a compoundthat is not bound to an antibody) is excited by linearly polarizedlight, its rotation is much faster than the correspondingtracer-antibody conjugate produced in a competitive binding immunoassay.FPIAs are advantageous over RIAs inasmuch as there are no radioactivesubstances requiring special handling and disposal. In addition, FPIAsare homogeneous assays that can be easily and rapidly performed.

In view of the above, a method of determining the presence, amount, orconcentration of analyte (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for an analyte(or a fragment thereof) by an assay (i) employing (i′) at least one ofan antibody, a fragment of an antibody that can bind to an analyte, avariant of an antibody that can bind to an analyte, a fragment of avariant of an antibody that can bind to an analyte, and a DVD-Ig (or afragment, a variant, or a fragment of a variant thereof) that can bindto an analyte, and (ii′) at least one detectable label and (ii)comprising comparing a signal generated by the detectable label as adirect or indirect indication of the presence, amount or concentrationof analyte (or a fragment thereof) in the test sample to a signalgenerated as a direct or indirect indication of the presence, amount orconcentration of analyte (or a fragment thereof) in a control orcalibrator. The calibrator is optionally part of a series ofcalibrators, in which each of the calibrators differs from the othercalibrators by the concentration of analyte.

The method can comprise (i) contacting the test sample with at least onefirst specific binding partner for analyte (or a fragment thereof)selected from the group consisting of an antibody, a fragment of anantibody that can bind to an analyte, a variant of an antibody that canbind to an analyte, a fragment of a variant of an antibody that can bindto an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment ofa variant thereof) that can bind to an analyte so as to form a firstspecific binding partner/analyte (or fragment thereof) complex, (ii)contacting the first specific binding partner/analyte (or fragmentthereof) complex with at least one second specific binding partner foranalyte (or fragment thereof) selected from the group consisting of adetectably labeled anti-analyte antibody, a detectably labeled fragmentof an anti-analyte antibody that can bind to analyte, a detectablylabeled variant of an anti-analyte antibody that can bind to analyte, adetectably labeled fragment of a variant of an anti-analyte antibodythat can bind to analyte, and a detectably labeled DVD-Ig (or afragment, a variant, or a fragment of a variant thereof) so as to form afirst specific binding partner/analyte (or fragment thereof)/secondspecific binding partner complex, and (iii) determining the presence,amount or concentration of analyte in the test sample by detecting ormeasuring the signal generated by the detectable label in the firstspecific binding partner/analyte (or fragment thereof)/second specificbinding partner complex formed in (ii). A method in which at least onefirst specific binding partner for analyte (or a fragment thereof)and/or at least one second specific binding partner for analyte (or afragment thereof) is a DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof) as described herein can be preferred.

Alternatively, the method can comprise contacting the test sample withat least one first specific binding partner for analyte (or a fragmentthereof) selected from the group consisting of an antibody, a fragmentof an antibody that can bind to an analyte, a variant of an antibodythat can bind to an analyte, a fragment of a variant of an antibody thatcan bind to an analyte, and a DVD-Ig (or a fragment, a variant, or afragment of a variant thereof) and simultaneously or sequentially, ineither order, contacting the test sample with at least one secondspecific binding partner, which can compete with analyte (or a fragmentthereof) for binding to the at least one first specific binding partnerand which is selected from the group consisting of a detectably labeledanalyte, a detectably labeled fragment of analyte that can bind to thefirst specific binding partner, a detectably labeled variant of analytethat can bind to the first specific binding partner, and a detectablylabeled fragment of a variant of analyte that can bind to the firstspecific binding partner. Any analyte (or a fragment thereof) present inthe test sample and the at least one second specific binding partnercompete with each other to form a first specific binding partner/analyte(or fragment thereof) complex and a first specific bindingpartner/second specific binding partner complex, respectively. Themethod further comprises determining the presence, amount orconcentration of analyte in the test sample by detecting or measuringthe signal generated by the detectable label in the first specificbinding partner/second specific binding partner complex formed in (ii),wherein the signal generated by the detectable label in the firstspecific binding partner/second specific binding partner complex isinversely proportional to the amount or concentration of analyte in thetest sample.

The above methods can further comprise diagnosing, prognosticating, orassessing the efficacy of a therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the efficacy of a therapeutic/prophylactic treatmentof the patient from whom the test sample was obtained, the methodoptionally further comprises modifying the therapeutic/prophylactictreatment of the patient as needed to improve efficacy. The method canbe adapted for use in an automated system or a semi-automated system.

With regard to the methods of assay (and kit therefor), it may bepossible to employ commercially available anti-analyte antibodies ormethods for production of anti-analyte as described in the literature.Commercial supplies of various antibodies include, but are not limitedto, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech,Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for analyteor a fragment thereof, e.g., for detecting disease or risk of disease.Generally, in making such a comparison, the predetermined level isobtained by running a particular assay a sufficient number of times andunder appropriate conditions such that a linkage or association ofanalyte presence, amount or concentration with a particular stage orendpoint of a disease, disorder or condition or with particular clinicalindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects). The analytemeasured can include fragments thereof, degradation products thereof,and/or enzymatic cleavage products thereof.

In particular, with respect to a predetermined level as employed formonitoring disease progression and/or treatment, the amount orconcentration of analyte or a fragment thereof may be “unchanged,”“favorable” (or “favorably altered”), or “unfavorable” (or “unfavorablyaltered”). “Elevated” or “increased” refers to an amount or aconcentration in a test sample that is higher than a typical or normallevel or range (e.g., predetermined level), or is higher than anotherreference level or range (e.g., earlier or baseline sample). The term“lowered” or “reduced” refers to an amount or a concentration in a testsample that is lower than a typical or normal level or range (e.g.,predetermined level), or is lower than another reference level or range(e.g., earlier or baseline sample). The term “altered” refers to anamount or a concentration in a sample that is altered (increased ordecreased) over a typical or normal level or range (e.g., predeterminedlevel), or over another reference level or range (e.g., earlier orbaseline sample).

The typical or normal level or range for analyte is defined inaccordance with standard practice. Because the levels of analyte in someinstances will be very low, a so-called altered level or alteration canbe considered to have occurred when there is any net change as comparedto the typical or normal level or range, or reference level or range,that cannot be explained by experimental error or sample variation.Thus, the level measured in a particular sample will be compared withthe level or range of levels determined in similar samples from aso-called normal subject. In this context, a “normal subject” is anindividual with no detectable disease, for example, and a “normal”(sometimes termed “control”) patient or population is/are one(s) thatexhibit(s) no detectable disease, respectively, for example.Furthermore, given that analyte is not routinely found at a high levelin the majority of the human population, a “normal subject” can beconsidered an individual with no substantial detectable increased orelevated amount or concentration of analyte, and a “normal” (sometimestermed “control”) patient or population is/are one(s) that exhibit(s) nosubstantial detectable increased or elevated amount or concentration ofanalyte. An “apparently normal subject” is one in which analyte has notyet been or currently is being assessed. The level of an analyte is saidto be “elevated” when the analyte is normally undetectable (e.g., thenormal level is zero, or within a range of from about 25 to about 75percentiles of normal populations), but is detected in a test sample, aswell as when the analyte is present in the test sample at a higher thannormal level. Thus, inter alia, the disclosure provides a method ofscreening for a subject having, or at risk of having, a particulardisease, disorder, or condition. The method of assay can also involvethe assay of other markers and the like.

Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing a givendisease, disorder or condition. Specifically, such a method can comprisethe steps of:

(a) determining the concentration or amount in a test sample from asubject of analyte (or a fragment thereof) (e.g., using the methodsdescribed herein, or methods known in the art); and

-   -   (b) comparing the concentration or amount of analyte (or a        fragment thereof) determined in step (a) with a predetermined        level, wherein, if the concentration or amount of analyte        determined in step (a) is favorable with respect to a        predetermined level, then the subject is determined not to have        or be at risk for a given disease, disorder or condition.        However, if the concentration or amount of analyte determined in        step (a) is unfavorable with respect to the predetermined level,        then the subject is determined to have or be at risk for a given        disease, disorder or condition.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of:

(a) determining the concentration or amount in a test sample from asubject of analyte;

(b) determining the concentration or amount in a later test sample fromthe subject of analyte; and

(c) comparing the concentration or amount of analyte as determined instep (b) with the concentration or amount of analyte determined in step(a), wherein if the concentration or amount determined in step (b) isunchanged or is unfavorable when compared to the concentration or amountof analyte determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened. By comparison, ifthe concentration or amount of analyte as determined in step (b) isfavorable when compared to the concentration or amount of analyte asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved.

Optionally, the method further comprises comparing the concentration oramount of analyte as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of analyteas determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

Still further, the methods can be used to monitor treatment in a subjectreceiving treatment with one or more pharmaceutical compositions.Specifically, such methods involve providing a first test sample from asubject before the subject has been administered one or morepharmaceutical compositions. Next, the concentration or amount in afirst test sample from a subject of analyte is determined (e.g., usingthe methods described herein or as known in the art). After theconcentration or amount of analyte is determined, optionally theconcentration or amount of analyte is then compared with a predeterminedlevel. If the concentration or amount of analyte as determined in thefirst test sample is lower than the predetermined level, then thesubject is not treated with one or more pharmaceutical compositions.However, if the concentration or amount of analyte as determined in thefirst test sample is higher than the predetermined level, then thesubject is treated with one or more pharmaceutical compositions for aperiod of time. The period of time that the subject is treated with theone or more pharmaceutical compositions can be determined by one skilledin the art (for example, the period of time can be from about seven (7)days to about two years, preferably from about fourteen (14) days toabout one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of analyte is determined in thesecond or subsequent test sample is determined (e.g., using the methodsdescribed herein or as known in the art). The concentration or amount ofanalyte as determined in each of the second and subsequent test samplesis then compared with the concentration or amount of analyte asdetermined in the first test sample (e.g., the test sample that wasoriginally optionally compared to the predetermined level). If theconcentration or amount of analyte as determined in step (c) isfavorable when compared to the concentration or amount of analyte asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved, and the subject shouldcontinue to be administered the one or pharmaceutical compositions ofstep (b). However, if the concentration or amount determined in step (c)is unchanged or is unfavorable when compared to the concentration oramount of analyte as determined in step (a), then the disease in thesubject is determined to have continued, progressed or worsened, and thesubject should be treated with a higher concentration of the one or morepharmaceutical compositions administered to the subject in step (b) orthe subject should be treated with one or more pharmaceuticalcompositions that are different from the one or more pharmaceuticalcompositions administered to the subject in step (b). Specifically, thesubject can be treated with one or more pharmaceutical compositions thatare different from the one or more pharmaceutical compositions that thesubject had previously received to decrease or lower said subject'sanalyte level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained.

When used to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3days, about 4 days, about 5 days, about 6 days or about 7 days), and theinitial assay likewise is generally done within a shorter timeframe,e.g., about minutes, hours or days of the onset of the disease orcondition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the above assays can be performed using a first test sampleobtained from a subject where the first test sample is obtained from onesource, such as urine, serum or plasma. Optionally, the above assays canthen be repeated using a second test sample obtained from the subjectwhere the second test sample is obtained from another source. Forexample, if the first test sample was obtained from urine, the secondtest sample can be obtained from serum or plasma. The results obtainedfrom the assays using the first test sample and the second test samplecan be compared. The comparison can be used to assess the status of adisease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from a given disease,disorder or condition will benefit from treatment. In particular, thedisclosure relates to analyte companion diagnostic methods and products.Thus, the method of “monitoring the treatment of disease in a subject”as described herein further optimally also can encompass selecting oridentifying candidates for therapy.

Thus, in particular embodiments, the disclosure also provides a methodof determining whether a subject having, or at risk for, a givendisease, disorder or condition is a candidate for therapy. Generally,the subject is one who has experienced some symptom of a given disease,disorder or condition or who has actually been diagnosed as having, orbeing at risk for, a given disease, disorder or condition, and/or whodemonstrates an unfavorable concentration or amount of analyte or afragment thereof, as described herein.

The method optionally comprises an assay as described herein, whereanalyte is assessed before and following treatment of a subject with oneor more pharmaceutical compositions (e.g., particularly with apharmaceutical related to a mechanism of action involving analyte), withimmunosuppressive therapy, or by immunoabsorption therapy, or whereanalyte is assessed following such treatment and the concentration orthe amount of analyte is compared against a predetermined level. Anunfavorable concentration of amount of analyte observed followingtreatment confirms that the subject will not benefit from receivingfurther or continued treatment, whereas a favorable concentration oramount of analyte observed following treatment confirms that the subjectwill benefit from receiving further or continued treatment. Thisconfirmation assists with management of clinical studies, and provisionof improved patient care.

It goes without saying that, while certain embodiments herein areadvantageous when employed to assess a given disease, disorder orcondition as discussed herein, the assays and kits can be employed toassess analyte in other diseases, disorders and conditions. The methodof assay can also involve the assay of other markers and the like.

The method of assay also can be used to identify a compound thatameliorates a given disease, disorder or condition. For example, a cellthat expresses analyte can be contacted with a candidate compound. Thelevel of expression of analyte in the cell contacted with the compoundcan be compared to that in a control cell using the method of assaydescribed herein.

II. Kit

A kit for assaying a test sample for the presence, amount orconcentration of an analyte (or a fragment thereof) in a test sample isalso provided. The kit comprises at least one component for assaying thetest sample for the analyte (or a fragment thereof) and instructions forassaying the test sample for the analyte (or a fragment thereof). The atleast one component for assaying the test sample for the analyte (or afragment thereof) can include a composition comprising an anti-analyteDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),which is optionally immobilized on a solid phase.

The kit can comprise at least one component for assaying the test samplefor an analyte by immunoassay, e.g., chemiluminescent microparticleimmunoassay, and instructions for assaying the test sample for ananalyte by immunoassay, e.g., chemiluminescent microparticleimmunoassay. For example, the kit can comprise at least one specificbinding partner for an analyte, such as an anti-analyte,monoclonal/polyclonal antibody (or a fragment thereof that can bind tothe analyte, a variant thereof that can bind to the analyte, or afragment of a variant that can bind to the analyte) or an anti-analyteDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),either of which can be detectably labeled. Alternatively oradditionally, the kit can comprise detectably labeled analyte (or afragment thereof that can bind to an anti-analyte, monoclonal/polyclonalantibody or an anti-analyte DVD-Ig (or a fragment, a variant, or afragment of a variant thereof)), which can compete with any analyte in atest sample for binding to an anti-analyte, monoclonal/polyclonalantibody (or a fragment thereof that can bind to the analyte, a variantthereof that can bind to the analyte, or a fragment of a variant thatcan bind to the analyte) or an anti-analyte DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof), either of which can beimmobilized on a solid support. The kit can comprise a calibrator orcontrol, e.g., isolated or purified analyte. The kit can comprise atleast one container (e.g., tube, microtiter plates or strips, which canbe already coated with a first specific binding partner, for example)for conducting the assay, and/or a buffer, such as an assay buffer or awash buffer, either one of which can be provided as a concentratedsolution, a substrate solution for the detectable label (e.g., anenzymatic label), or a stop solution. Preferably, the kit comprises allcomponents, i.e., reagents, standards, buffers, diluents, etc., whichare necessary to perform the assay. The instructions can be in paperform or computer-readable form, such as a disk, CD, DVD, or the like.

Any antibodies, such as an anti-analyte antibody or an anti-analyteDVD-Ig, or tracer can incorporate a detectable label as describedherein, such as a fluorophore, a radioactive moiety, an enzyme, abiotin/avidin label, a chromophore, a chemiluminescent label, or thelike, or the kit can include reagents for carrying out detectablelabeling. The antibodies, calibrators and/or controls can be provided inseparate containers or pre-dispensed into an appropriate assay format,for example, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, enzyme substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instruments for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, a solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip.

III. Adaptation of Kit and Method

The kit (or components thereof), as well as the method of determiningthe presence, amount or concentration of an analyte in a test sample byan assay, such as an immunoassay as described herein, can be adapted foruse in a variety of automated and semi-automated systems (includingthose wherein the solid phase comprises a microparticle), as described,e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commerciallymarketed, e.g., by Abbott Laboratories (Abbott Park, IL) as ARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., an anti-analyte,monoclonal/polyclonal antibody (or a fragment thereof, a variantthereof, or a fragment of a variant thereof) or an anti-analyte DVD-Ig(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) is attached; either way, sandwich formation and analytereactivity can be impacted), and the length and timing of the capture,detection and/or any optional wash steps. Whereas a non-automatedformat, such as an ELISA, may require a relatively longer incubationtime with sample and capture reagent (e.g., about 2 hours), an automatedor semi-automated format (e.g., ARCHITECT®, Abbott Laboratories) mayhave a relatively shorter incubation time (e.g., approximately 18minutes for ARCHITECT®). Similarly, whereas a non-automated format, suchas an ELISA, may incubate a detection antibody, such as the conjugatereagent, for a relatively longer incubation time (e.g., about 2 hours),an automated or semi-automated format (e.g., ARCHITECT®) may have arelatively shorter incubation time (e.g., approximately 4 minutes forthe ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays. Immunosensorsand their methods of manufacture and operation in single-use testdevices are described, for example in, U.S. Pat. No. 5,063,081, U.S.Pat. App. Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577,U.S. Pat. App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No.2006/0160164, which are incorporated in their entireties by referencefor their teachings regarding same.

In particular, with regard to the adaptation of an analyte assay to theI-STAT® system, the following configuration is preferred. Amicrofabricated silicon chip is manufactured with a pair of goldamperometric working electrodes and a silver-silver chloride referenceelectrode. On one of the working electrodes, polystyrene beads (0.2 mmdiameter) with immobilized anti-analyte, monoclonal/polyclonal antibody(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) or anti-analyte DVD-Ig (or a fragment thereof, a variantthereof, or a fragment of a variant thereof), are adhered to a polymercoating of patterned polyvinyl alcohol over the electrode. This chip isassembled into an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising a specific binding partner foran analyte, such as an anti-analyte, monoclonal/polyclonal antibody (ora fragment thereof, a variant thereof, or a fragment of a variantthereof that can bind the analyte) or an anti-analyte DVD-Ig (or afragment thereof, a variant thereof, or a fragment of a variant thereofthat can bind the analyte), either of which can be detectably labeled.Within the fluid pouch of the cartridge is an aqueous reagent thatincludes p-aminophenol phosphate.

In operation, a sample suspected of containing an analyte is added tothe holding chamber of the test cartridge, and the cartridge is insertedinto the I-STAT® reader. After the specific binding partner for ananalyte has dissolved into the sample, a pump element within thecartridge forces the sample into a conduit containing the chip. Here itis oscillated to promote formation of the sandwich. In the penultimatestep of the assay, fluid is forced out of the pouch and into the conduitto wash the sample off the chip and into a waste chamber. In the finalstep of the assay, the alkaline phosphatase label reacts withp-aminophenol phosphate to cleave the phosphate group and permit theliberated p-aminophenol to be electrochemically oxidized at the workingelectrode. Based on the measured current, the reader is able tocalculate the amount of analyte in the sample by means of an embeddedalgorithm and factory-determined calibration curve.

It further goes without saying that the methods and kits as describedherein necessarily encompass other reagents and methods for carrying outthe immunoassay. For instance, encompassed are various buffers such asare known in the art and/or which can be readily prepared or optimizedto be employed, e.g., for washing, as a conjugate diluent, microparticlediluent, and/or as a calibrator diluent. An exemplary conjugate diluentis ARCHITECT® conjugate diluent employed in certain kits (AbbottLaboratories, Abbott Park, IL) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, IL), which comprises a buffer containing MES,other salt, a protein blocker, and an antimicrobial agent. Additionally,as described in U.S. Patent Application No. 61/142,048 filed Dec. 31,2008, improved signal generation may be obtained, e.g., in an I-Statcartridge format, using a nucleic acid sequence linked to the signalantibody as a signal amplifier.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXEMPLIFICATION Example 1 Design, Construction, and Analysis of a DVD-IgExample 1.1 Assays Used to Identify and Characterize Parent Antibodiesand DVD-Ig

The following assays were used throughout the Examples to identify andcharacterize parent antibodies and DVD-Ig, unless otherwise stated.

Example 1.1.1 Assays Used To Determine Binding and Affinity of ParentAntibodies and DVD-Ig for Their Target Antigen(s) Example 1.1.1.A DirectBind ELISA

Enzyme Linked Immunosorbent Assays to screen for antibodies that bind adesired target antigen were performed as follows. High bind ELISA plates(Corning Costar #3369, Acton, Mass.) were coated with 100 μL/well of 10μg/ml of desired target antigen (R&D Systems, Minneapolis, Minn.) ordesired target antigen extra-cellular domain/FC fusion protein (R&DSystems, Minneapolis, Minn.) or monoclonal mouse anti-polyHistidineantibody (R&D Systems # MAB050, Minneapolis, Minn.) in phosphatebuffered saline (10×PBS, Abbott Bioresearch Center, Media Prep# MPS-073,Worcester, Mass.) overnight at 4° C. Plates were washed four times withPBS containing 0.02% Tween 20. Plates were blocked by the addition of300 μL/well blocking solution (non-fat dry milk powder, various retailsuppliers, diluted to 2% in PBS) for 1/2 hour at room temperature.Plates were washed four times after blocking with PBS containing 0.02%Tween 20.

Alternatively, one hundred microliters per well of 10 μg/ml of Histidine(His) tagged desired target antigen (R&D Systems, Minneapolis, Minn.)was added to ELISA plates coated with monoclonal mouseanti-polyHistidine antibody as described above and incubated for 1 hourat room temperature. Wells were washed four times with PBS containing0.02% Tween 20.

One hundred microliters of antibody or DVD-Ig preparations diluted inblocking solution as described above was added to the desired targetantigen plate or desired target antigen/FC fusion plate or theanti-polyHistidine antibody/His tagged desired target antigen plateprepared as described above and incubated for 1 hour at roomtemperature. Wells were washed four times with PBS containing 0.02%Tween 20.

One hundred microliters of 10 ng/mL goat anti-human IgG-FC specific HRPconjugated antibody (Southern Biotech #2040-05, Birmingham, Ala.) wasadded to each well of the desired target antigen plate oranti-polyHistidine antibody/Histidine tagged desired target antigenplate. Alternatively, one hundred microliters of 10 ng/mL goatanti-human IgG-kappa light chain specific HRP conjugated antibody(Southern Biotech #2060-05 Birmingham, Ala.) was added to each well ofthe desired target antigen/FC fusion plate and incubated for 1 hour atroom temperature. Plates were washed 4 times with PBS containing 0.02%Tween 20.

One hundred microliters of enhanced TMB solution (Neogen Corp. #308177,K Blue, Lexington, Ky.) was added to each well and incubated for 10minutes at room temperature. The reaction was stopped by the addition of50 μL 1N sulphuric acid. Plates were read spectrophotometrically at awavelength of 450 nm.

Table 3 contains a list of the antigens used in the Direct Bind Assay.

Table 4 contains the binding data expressed as EC50 in nM for thoseantibodies and DVD-Ig constructs tested in the Direct Bind ELISA assay.

In the Direct Bind ELISA, binding was sometimes not observed, probablybecause the antibody binding site on the target antigen was either“masked” or the antigen is “distorted” when coated to the plasticsurface. The inability of a DVD-Ig to bind its target may also be due tosteric limitation imposed on DVD-Ig by the Direct Bind ELISA format. Theparent antibodies and DVD-Igs that did not bind in the Direct Bind ELISAformat bound to target antigen in other ELISA formats, such as FACS,Biacore or bioassay. Non-binding of a DVD-Ig was also restored byadjusting the linker length between the two variable domains of theDVD-Ig, as shown previously.

TABLE 3 Antigens Used in Direct Bind ELISA Assay Antigen VendorDesignation Vendor Catalog # RGMa RGM-A Repulsive Guidence R&D 2459-RMMolecule A RAGE RAGE RAGE/FC Chimera R&D 1145-RG IL-18 IL-18 IL-18 MBLB003-5 IGF1 IGF-1 IGF-I R&D 291-G1 IGF2 IGF-2 IGF-II R&D 292-G2 IL-1βIL-1β IL-1β R&D 201-LB TNFα TNFα TNFα/TNFSF1A R&D 210-TA EGFR EGFR EGFR/FC Chimera R&D 1095-ER

/FC=Fc Region Fusion

TABLE 4 Direct Binding ELISA of Parental Antibodies and DVD ConstructsParent Direct Bind Direct Bind Antibody N-Terminal C-Terminal ELISA N-ELISA C- or DVD Variable Variable Terminal VD Terminal VD ID DomainDomain EC50 (nM) EC50 (nM) AB017 TNF 0.44 DVD223 A beta (seq. 1) TNF8.59 DVD224 TNF A beta (seq. 1) 0.43 AB032 IL-1b (seq. 1) 5.27 DVD225 Abeta (seq. 1) IL-1b (seq. 1) 3938.56 DVD226 IL-1b (seq. 1) A beta(seq. 1) 9.52 AB010 IGF1, 2 10.1, 2.93 DVD229 A beta (seq. 1) IGF1, 2210.16, 130.87 DVD230 IGF1, 2 A beta (seq. 1) 4.84, 2.44 AB058 IL-18(seq. 1) 3.77 DVD231 A beta (seq. 1) IL-18 (seq. 1) 50.62 DVD232 IL-18(seq. 1) A beta (seq. 1) 0.68 AB033 EGFR (seq. 1) 0.68 DVD236 EGFR(seq. 1) CD-20 (seq. 1) 0.49 AB017 TNF 0.19 AB033 EGFR (seq. 1) 0.63DVD289 TNF EGFR (seq. 1) 0.32 3.65 DVD290 EGFR (seq. 1) TNF 0.54 41.05AB017 TNF 0.19 AB059 RGMa 0.13 DVD291 TNF RGMa 0.17 0.98 DVD292 RGMa TNF2.16 775.32 AB060 RAGE (seq. 1) 1.69 DVD293 RAGE (seq. 1) A beta(seq. 1) 7.32 DVD294 A beta (seq. 1) RAGE (seq. 1) 1.53 AB060 RAGE(seq. 1) 0.64 AB017 TNF 0.50 DVD295 RAGE (seq. 1) TNF 0.48 6.57 DVD296TNF RAGE (seq. 1) 0.33 1.05

Binding of all DVD constructs was maintained and comparable to parentantibodies. All N-terminal variable domains bound with a similar highaffinity as the parent antibody as well as the C-terminal variabledomains of DVD constructs DVD289, DVD291, DVD294 and DVD296.

Example 1.1.1.B Aβ 1-42 ELISA

ELISA assays were performed to screen for antibodies that bind a desiredtarget antigen as follows. Maxisorb NUNC Immuno plates were coated with100 μL/well of 1 μg/ml of Aβ1-42 diluted in coating buffer overnight at6° C. Plates were washed three times with PBS containing 0.02% Tween 20.Blocking solution was prepared by dissolving the blocking reagent forELISA (Roche) in 100 mL water and then diluted further 1:10 prior touse. Plates were blocked by the addition of 265 μL/well blockingsolution for 2 hours at room temperature. Plates were washed three timesafter blocking with PBS containing 0.02% Tween 20.

Antibody or DVD-Igs were diluted to 100 ng/μL in PBST+0.5% BSA andserially diluted 1:3. One hundred microliters of antibody or DVD-Igpreparations was added to the plate and incubated for 2 hour at roomtemperature. Wells were washed three times with PBS containing 0.02%Tween 20.

Two hundred microliters of anti-human POD diluted 1:5000 was added toeach well of the plate and incubated for 1 hour at room temperature.Plates were washed three times with PBS containing 0.02% Tween 20.

One hundred microliters of TMB solution was added to each well andincubated for 10 minutes. TMB solution was prepared by mixing 20 mL 100mM natrium-acetate pH4.9 with 200 μL TMB solution (Roche #92817060) and29.5 μL 3% H₂O₂. The reaction was stopped by the addition of 50 μL 1Nsulphuric acid. Plates were read spectrophotometrically at a wavelengthof 450 nm. Results are provided in Table 5.

TABLE 5 Aβ 1-42 ELISA of Parental Antibodies and DVD Constructs ParentAβ Aβ Antibody N-Terminal C-Terminal ELISA N- ELISA C- or DVD VariableVariable Terminal VD Terminal VD ID Domain Domain EC50 (nM) EC50 (nM)AB043 A beta (seq. 1) 0.012 DVD223 A beta (seq. 1) TNF 0.014 DVD224 TNFA beta (seq. 1) 0.032 AB043 A beta (seq. 1) 0.012 DVD225 A beta (seq. 1)IL-1b (seq. 1) 0.016 DVD226 IL-1b (seq. 1) A beta (seq. 1) 0.045 AB043 Abeta (seq. 1) 0.012 DVD229 A beta (seq. 1) IGF1, 2 0.016 DVD230 IGF1, 2A beta (seq. 1) 0.039 AB043 A beta (seq. 1) 0.012 DVD231 A beta (seq. 1)IL-18 (seq. 1) 0.014 DVD232 IL-18 (seq. 1) A beta (seq. 1) 0.048 AB043 Abeta (seq. 1) 0.012 DVD233 A beta (seq. 1) IL-6 0.013 AB043 A beta(seq. 1) 0.012 DVD293 RAGE (seq. 1) A beta (seq. 1) 0.048 DVD294 A beta(seq. 1) RAGE (seq. 1) 0.014 AB043 A beta (seq. 1) 0.012 DVD201 7C6 Abeta (seq. 1) 0.024 DVD202 A beta (seq. 1) 7C6 0.017

Binding of all DVD constructs was maintained and comparable to parentantibodies. All N-terminal variable domains bound with a similar highaffinity as the parent antibody.

Example 1.1.1.C Aβ 20-42 ELISA

ELISA assays were performed to screen for antibodies that bind a desiredtarget antigen as follows. Aβ 20-42 globulomer and PBS at 37° C. waspre-warmed for 1 minute. High bind ELISA plates (Corning Costar #3369,Acton, Mass.) were coated with 100 μL/well of 41 g/ml of Aβ 20-42globulomer diluted in PBS overnight at 4° C. Plates were blocked with320 μL/well of 100% superblock PBS buffer (Thermo Scientific, #37515)and incubated for 45 minutes at room temperature. Plates were washedthree times after blocking with PBS containing 0.02% Tween 20.

Antibody or DVD-Igs were diluted to 2000 ng/mL, 1000 ng/mL, 200 ng/mL,40 ng/mL, 8 ng/mL, 1.6 ng/mL and 0.32 ng/mL in 10% superblock in PBS.One hundred microliters of antibody or DVD-Ig preparations was added tothe plate in triplicate and incubated for 2 hours at room temperature.Wells were washed five times with PBS containing 0.02% Tween 20.

One hundred microliters of goat anti-human HRP conjugated antibody(Pierce #31412) diluted 1:40,000 was added to each well of the plate andincubated for 30 minutes at room temperature. Plates were washed fivetimes with PBS containing 0.02% Tween 20.

One hundred microliters of TMB solution (Invitrogen-Zymed; #00-2023) wasadded to each well and incubated for 5 minutes. The reaction was stoppedby the addition of 100 μL 2N sulphuric acid. Plates were readspectrophotometrically at a wavelength of 450 nm. Results are shown inTable 6.

TABLE 6 Aβ 20-42 ELISA of Parental Antibodies and DVD Constructs ParentAβ Aβ Antibody N-Terminal C-Terminal ELISA N- ELISA C- or DVD VariableVariable Terminal VD Terminal VD ID Domain Domain EC50 (nM) EC50 (nM)AB044 A beta (seq 2) 0.319 DVD201 A beta (seq 2) A beta (seq 1) 0.397DVD202 A beta (seq 1) A beta (seq 2) 0.178

Binding of all DVD constructs was maintained and was comparable toparent antibodies.

Example 1.1.1.D Capture ELISA

ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) are incubated overnightat 4° C. with anti-human Fc antibody (5 μg/ml in PBS, JacksonImmunoresearch, West Grove, Pa.). Plates are washed three times inwashing buffer (PBS containing 0.05% Tween 20), and blocked for 1 hourat 25° C. in blocking buffer (PBS containing 1% BSA). Wells are washedthree times, and serial dilutions of each antibody or DVD-Ig in PBScontaining 0.1% BSA are added to the wells and incubated at 25° C. for 1hour. The wells are washed three times, and biotinylated antigen (2 nM)is added to the plates and incubated for 1 hour at 25° C. The wells arewashed three times and incubated for 1 hour at 25° C. withstreptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The wells arewashed three times, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford,Ill.) is added per well. Following color development the reaction isstopped with 1N HCL and absorbance at 450 nM is measured. Results areshown in Table 7.

Example 1.1.1.E Affinity Determination Using BIACORE Technology

TABLE 7 Reagent Used in Biacore Analyses Assay Antigen VendorDesignation Vendor Catalog # IL-1β Recombinant Human R&D systems 201-LBIL-1β TNFα Recombinant Human R&D systems 210-TA TNF-α/TNFSF1A EGFRRecombinant Human R&D systems 1095-ER EGF Receptor

ECD=Extracellular Domain

/FC=antigen/IgG FC domain fusion protein

BIACORE Methods:

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines theaffinity of antibodies or DVD-Ig with kinetic measurements of on-rateand off-rate constants. Binding of antibodies or DVD-Ig to a targetantigen (for example, a purified recombinant target antigen) isdetermined by surface plasmon resonance-based measurements with aBiacore® 1000 or 3000 instrument (Biacore® AB, Uppsala, Sweden) usingrunning HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%surfactant P20) at 25° C. All chemicals are obtained from Biacore® AB(Uppsala, Sweden) or otherwise from a different source as described inthe text. For example, approximately 5000 RU of goat anti-mouse IgG,(Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc,Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) is directlyimmobilized across a CMS research grade biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface areblocked with ethanolamine. Modified carboxymethyl dextran surface inflowcell 2 and 4 is used as a reaction surface. Unmodified carboxymethyldextran without goat anti-mouse IgG in flow cell 1 and 3 is used as thereference surface. For kinetic analysis, rate equations derived from the1:1 Langmuir binding model are fitted simultaneously to association anddissociation phases of all eight injections (using global fit analysis)with the use of Biaevaluation 4.0.1 software. Purified antibodies orDVD-Ig are diluted in HEPES-buffered saline for capture across goatanti-mouse IgG specific reaction surfaces. Antibodies or DVD-Ig to becaptured as a ligand (25 μg/ml) are injected over reaction matrices at aflow rate of 5 μl/min. The association and dissociation rate constants,k_(m) (M⁻¹ s⁻¹) and k_(off) (s⁻¹) are determined under a continuous flowrate of 25 μl/min. Rate constants are derived by making kinetic bindingmeasurements at different antigen concentrations ranging from 10-200 nM.The equilibrium dissociation constant (M) of the reaction betweenantibodies or DVD-Igs and the target antigen is then calculated from thekinetic rate constants by the following formula: K_(D)=k_(off)/k_(on).Binding is recorded as a function of time and kinetic rate constants arecalculated. In this assay, on-rates as fast as 10⁶M⁻¹ s⁻¹ and off-ratesas slow as 10⁻⁶ can can be measured. Results are shown in Table 8.

TABLE 8 BIACORE Analysis of Parental Anti- bodies and DVD ConstructsParent  N-Ter- C-Ter- Anti- minal minal body Variable Variable k_(on)or DVD- Domain Domain (M-1s- k_(off) K_(D) Ig ID (VD) (VD) 1) (s-1) (M)AB017 TNF 3.37E+ 9.57E− 2.84E− 06 05 11 DVD223 TNF 3.80E+ 1.04E− 2.74E−04 04 09 DVD224 TNF 1.03E+ 8.60E− 8.36E− 06 05 11 AB017 TNF 3.37E+9.57E− 2.84E− 06 05 11 AB033 EGFR 7.81E+ 1.30E− 1.67E− (SEQ1) 05 03 09DVD289 TNF 1.25E+ 8.82E− 7.05E− 06 05 11 DVD289 EGFR 8.73E+ 6.77E−7.76E−  (SEQ1) 04 04 09 DVD290 EGFR 1.06E+ 1.31E− 1.23E− (SEQ1) 06 03 09DVD290 TNF 1.74E+ 1.45E− 8.31E− 06 04 11 AB017 TNF 3.37E+ 9.57E− 2.84E−06 05 11 AB059 RGMA 9.52E+ 6.09E− 6.40E− 05 04 10 DVD291 TNF 1.74E+1.45E− 8.31E− 06 04 11 DVD291 RGMA 3.89E+ 5.04E− 1.30E− 04 04 08 DVD292RGMA 1.72E+ 5.23E− 3.04E− 06 04 10 DVD292 TNF 5.66E+ 1.25E− 2.21E− 04 0409 AB017 TNF 3.37E+ 9.57E− 2.84E− 06 05 11 DVD295 TNF 3.90E+ 1.04E−2.68E− 05 03 09 DVD296 TNF 1.35E+ 7.40E− 5.48E 06 05 −11

Binding of all DVD-Ig constructs characterized by Biacore technology wasmaintained and comparable to that of parent antibodies. All N-terminalvariable domains bound with a similar high affinity as the parentantibody.

Example 1.1.2 Assays Used to Determine the Functional Activity of ParentAntibodies and DVD-Ig Example 1.1.2.A Cytokine Bioassay

The ability of an anti-cytokine or an anti-growth factor parent antibodyor DVD-Ig containing anti-cytokine or anti-growth factor sequences toinhibit or neutralize a target cytokine or growth factor bioactivity isanalyzed by determining the inhibitory potential of the antibody orDVD-Ig. For example, the ability of an anti-IL-4 antibody to inhibitIL-4 mediated IgE production may be used. For example, human naive Bcells are isolated from peripheral blood, respectively, buffy coats byFicoll-paque density centrifugation, followed by magnetic separationwith MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) specificfor human sIgD FITC labeled goat F(ab)2 antibodies followed by anti-FITCMACS beads. Magnetically sorted naive B cells are adjusted to 3×10⁵cells per ml in XV15 and plated out in 100 μl per well of 96-well platesin a 6×6 array in the center of the plate, surrounded by PBS filledwells during the 10 days of culture at 37° C. in the presence of 5% CO₂.One plate each is prepared per antibody to be tested, consisting of 3wells each of un-induced and induced controls and quintuplicate repeatsof antibody titrations starting at 7 μg/ml and running in 3-folddilution down to 29 ng/ml final concentrations added in 50 μl four timesconcentrated pre-dilution. To induce IgE production, rhIL-4 at 20 ng/mlplus anti-CD40 monoclonal antibody (Novartis, Basel, Switzerland) at 0.5μg/ml final concentrations in 50 μl each are added to each well, and IgEconcentrations are determined at the end of the culture period by astandard sandwich ELISA method.

Example 1.1.2.B Cytokine Release Assay

The ability of a parent antibody or DVD-Ig to cause cytokine release isanalyzed. Peripheral blood is withdrawn from three healthy donors byvenipuncture into heparized vacutainer tubes. Whole blood is diluted 1:5with RPMI-1640 medium and placed in 24-well tissue culture plates at 0.5mL per well. The anti-cytokine antibodies (e.g., anti-IL-4) are dilutedinto RPMI-1640 and placed in the plates at 0.5 mL/well to give finalconcentrations of 200, 100, 50, 10, and 1 μg/mL. The final dilution ofwhole blood in the culture plates is 1:10. LPS and PHA are added toseparate wells at 2 μg/mL and 5 μg/mL final concentration as a positivecontrol for cytokine release. Polyclonal human IgG is used as negativecontrol antibody. The experiment is performed in duplicate. Plates areincubated at 37° C. at 5% CO₂. Twenty-four hours later the contents ofthe wells are transferred into test tubes and spun for 5 minutes at 1200rpm. Cell-free supernatants are collected and frozen for cytokineassays. Cells left over on the plates and in the tubes are lysed with0.5 mL of lysis solution, and placed at −20° C. and thawed. 0.5 mL ofmedium is added (to bring the volume to the same level as the cell-freesupernatant samples) and the cell preparations are collected and frozenfor cytokine assays. Cell-free supernatants and cell lysates are assayedfor cytokine levels by ELISA, for example, for levels of IL-8, IL-6,IL-1β, IL-IRA, or TNF-α.

Example 1.1.2.C Cytokine Cross-Reactivity Study

The ability of an anti-cytokine parent antibody or DVD-Ig directed to acytokine(s) of interest to cross react with other cytokines is analyzed.Parent antibodies or DVD-Ig are immobilized on a Biacore biosensormatrix. An anti-human Fc mAb is covalently linked via free amine groupsto the dextran matrix by first activating carboxyl groups on the matrixwith 100 mM N-hydroxysuccinimide (NHS) and 400 mMN-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).Approximately 50 μL of each antibody or DVD-Ig preparation at aconcentration of 25 μg/mL, diluted in sodium acetate, pH 4.5, isinjected across the activated biosensor and free amines on the proteinare bound directly to the activated carboxyl groups. Typically, 5000Resonance Units (RU's) are immobilized. Unreacted matrix EDC-esters aredeactivated by an injection of 1 M ethanolamine. A second flow cell isprepared as a reference standard by immobilizing human IgG1/K using thestandard amine coupling kit. SPR measurements are performed using the CMbiosensor chip. All antigens to be analyzed on the biosensor surface arediluted in HBS-EP running buffer containing 0.01% P20.

To examine the cytokine binding specificity, excess cytokine of interest(100 nM, e.g., soluble recombinant human) is injected across theanti-cytokine parent antibody or DVD-Ig immobilized biosensor surface (5minute contact time). Before injection of the cytokine of interest andimmediately afterward, HBS-EP buffer alone flows through each flow cell.The net difference in the signals between the baseline and the pointcorresponding to approximately 30 seconds after completion of cytokineinjection are taken to represent the final binding value. Again, theresponse is measured in Resonance Units. Biosensor matrices areregenerated using 10 mM HCl before injection of the next sample where abinding event is observed, otherwise running buffer was injected overthe matrices. Human cytokines (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-22, IL-23, IL-27, TNF-α, TNF-β, andIFN-γ, for example) are also simultaneously injected over theimmobilized mouse IgG1/K reference surface to record any nonspecificbinding background. By preparing a reference and reaction surface,Biacore can automatically subtract the reference surface data from thereaction surface data in order to eliminate the majority of therefractive index change and injection noise. Thus, it is possible toascertain the true binding response attributed to an anti-cytokineantibody or DVD-Ig binding reaction.

When a cytokine of interest is injected across immobilized anti-cytokineantibody, significant binding is observed. 10 mM HCl regenerationcompletely removes all non-covalently associated proteins. Examinationof the sensorgram shows that immobilized anti-cytokine antibody orDVD-Ig binding to soluble cytokine is strong and robust. Afterconfirming the expected result with the cytokine of interest, the panelof remaining recombinant human cytokines is tested, for each antibody orDVD-Ig separately. The amount of anti-cytokine antibody or DVD-Ig boundor unbound cytokine for each injection cycle is recorded. The resultsfrom three independent experiments are used to determine the specificityprofile of each antibody or DVD-1 g. Antibodies or DVD-Ig with theexpected binding to the cytokine of interest and no binding to any othercytokine are selected.

Example 1.1.2.D Tissue Cross Reactivity

Tissue cross reactivity studies are done in three stages, with the firststage including cryosections of 32 tissues, second stage including up to38 tissues, and the 3^(rd) stage including additional tissues from 3unrelated adults as described below. Studies are done typically at twodose levels.

Stage 1:

Cryosections (about 5 μm) of human tissues (32 tissues (typically:Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder, Heart,Skeletal Muscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, SpinalCord, Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, CerebralCortex, Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium,Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube andPlacenta) from one human donor obtained at autopsy or biopsy) are fixedand dried on object glass. The peroxidase staining of tissue sections isperformed, using the avidin-biotin system.

Stage 2:

Cryosections (about 5 μm) of human tissues 38 tissues (includingadrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix,esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node,breast mammary gland, ovary, oviduct, pancreas, parathyroid, peripheralnerve, pituitary, placenta, prostate, salivary gland, skin, smallintestine, spinal cord, spleen, stomach, striated muscle, testis,thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from 3unrelated adults obtained at autopsy or biopsy) are fixed and dried onobject glass. The peroxidase staining of tissue sections is performed,using the avidin-biotin system.

Stage 3:

Cryosections (about 5 μm) of cynomolgus monkey tissues (38 tissues(including adrenal, blood, blood vessel, bone marrow, cerebellum,cerebrum, cervix, esophagus, eye, heart, kidney, large intestine, liver,lung, lymph node, breast mammary gland, ovary, oviduct, pancreas,parathyroid, peripheral nerve, pituitary, placenta, prostate, salivarygland, skin, small intestine, spinal cord, spleen, stomach, striatedmuscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, anduterus) from 3 unrelated adult monkeys obtained at autopsy or biopsy)are fixed and dried on object glass. The peroxidase staining of tissuesections is performed, using the avidin-biotin system.

The antibody or DVD-Ig is incubated with the secondary biotinylatedanti-human IgG and developed into immune complex. The immune complex atthe final concentrations of 2 and 10 μg/mL of antibody or DVD-Ig isadded onto tissue sections on object glass and then the tissue sectionsare reacted for 30 minutes with a avidin-biotin-peroxidase kit.Subsequently, DAB (3,3′-diaminobenzidine), a substrate for theperoxidase reaction, is applied for 4 minutes for tissue staining.Antigen-Sepharose beads are used as positive control tissue sections.Target antigen and human serum blocking studies serve as additionalcontrols. The immune complex at the final concentrations of 2 and 10μg/mL of antibody or DVD-Ig is pre-incubated with target antigen (finalconcentration of 100 μg/ml) or human serum (final concentration 10%) for30 minutes, and then added onto the tissue sections on object glass andthen the tissue sections are reacted for 30 minutes with aavidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine),a substrate for the peroxidase reaction, is applied for 4 minutes fortissue staining.

Any specific staining is judged to be either an expected (e.g.,consistent with antigen expression) or unexpected reactivity based uponknown expression of the target antigen in question. Any staining judgedspecific is scored for intensity and frequency. The tissue stainingbetween stage 2 (human tissue) and stage 3 (cynomolgus monkey tissue) iseither judged to be similar or different.

Example 1.1.2.E Inhibition of HUVEC Proliferation/Survival by VEGFParent Antibody and DVD-Ig Constructs

Prior to plating for the assay, normal human umbilical vascularendothelial cells or HUVEC (passage 2-6) are maintained in EBM-2(Lonza-Clonetics, Walkersville, Md.) supplemented with EGM-2 SingleQuots(Lonza-Clonetics, Walkersville, Md., #CC-4176). HUVEC cells are platedat 10,000 cells/well on collagen coated black 96-well plates in (1000EMB-2 with 0.1% FBS in the absence of growth factors. The following daythe media is replaced with 0.1% FBS in the absence of growth factors.The following day the media is replaced with 100 μl of EMB-2 (withoutgrowth factors or serum) and incubated for four hours prior to theaddition of VEGF and antibodies/DVD-Igs. Anti-VEGF monoclonal antibodiesor DVD-Igs (at final concentrations of 67 nM, 6.7 nM and 0.67 nM) arediluted in EMB-2 with 0.1% BSA and are pre-incubated with recombinanthuman VEGF₁₆₅ (50 ng/ml) for 1 hour at 25° C. in 504 Antibody/DVD-Ig andVEGF mixtures are then added to the cells (500, and the plates wereincubated at 37° C. in a humidified, 5% CO₂ atmosphere for 72 hours.Cell survival/proliferation is measured indirectly by assessing ATPlevels using an ATPlite kit (Perkin Elmer, Waltham, Mass.) according tothe manufacturer's instructions.

Example 1.1.2.F IL-1α/β Bioassay and Neutralization Assay

MRCS cells were plated at 1.5-2×10⁴ cells per well in a 100 μL volumeand incubated overnight at 37° C., 5% CO₂. A 20 μg/mL working stock ofantibody (4× concentrated) was prepared in complete MEM medium. An eightpoint serial dilution was performed (5 μg/mL-0.0003 μg/mL) in completeMEM in Marsh dilution plates. Sixty-five μL/well of each antibodydilution was added in quadruplicate to a 96 well v-bottom (Costar#3894)plate and 65 μL of a 200 pg/mL solution of IL-1α or IL-1β or 65 μL of amixed solution containing a 50 pg/mL solution of both IL-1α and IL-1βwas also added. Control wells received 65 μL 200 pg/ml of IL-1α or IL-1βor 50 pg/mL mixed IL-1α/β (4× concentrated) plus 65 μL MEM media andmedia control wells received 130 μL of media. Following a 1 hourincubation, 100 μL of the Ab/Ag mixture was added to the MRCS cells. Allwell volumes were equal to 200 μL. All plate reagents were then 1×concentrated. After a 16-20 hour incubation, the well contents (150 μL)were transferred into a 96-well round bottom plate (Costar#3799) andplaced in a −20° C. freezer. The supernatants were tested for hIL-8levels by using a human IL-8 ELISA kit (R&D Systems, Minneapolis, Minn.)or hIL-8 chemiluminescence kit (MDS). Neutralization potency wasdetermined by calculating percent inhibition relative to the IL-1α,IL-1β, or the IL-1α/β alone control value. Results are shown in Table 9.

TABLE 9 IL-1βNeutralization Assay With IL-1β Parent Antibody and DVD-IgConstructs N-Terminal C-Terminal VD VD Parent N-Terminal C-TerminalIL-1β Neu- IL-1β Neu- Antibody Variable Variable tralization tralizationor DVD- Domain Domain Assay EC50 AssayEC50 Ig ID (VD) (VD) nM nM AB032IL-1β 0.05 DVD225 A beta (seq. 1) IL-1β 12.74 DVD226 IL-1β A beta(seq. 1) 0.028

All DVD-Igs containing VDs from AB032 in either the N-terminal orC-terminal position showed neutralization in the MRCS IL-11α/βneutralization assay.

Example 1.1.2.G Neutralization of huTNFα

L929 cells were grown to a semi-confluent density and harvested using0.05% tryspin (Gibco#25300). The cells were washed with PBS, counted andresuspended at 1E6 cells/mL in assay media containing 4 μg/mLactinomycin D. The cells were seeded in a 96-well plate (Costar#3599) ata volume of 50 μL and 5E4 cells/well. The DVD-Ig™ and control IgG werediluted to a 4× concentration in assay media and serial 1:3 dilutionswere performed. The huTNFα was diluted to 400 μg/mL in assay media.Antibody sample (200 μL) was added to the huTNFα (200 μL) in a 1:2dilution scheme and allowed to incubate for 0.5 hour at roomtemperature.

The DVD-Ig™/huTNFα solution was added to the plated cells at 100 μL fora final concentration of 100 pg/mL huTNFα and 25 nM-0.00014 nM DVD-Ig™.The plates were incubated for 20 hour at 37° C., 5% CO₂. To quantitateviability, 100 μL was removed from the wells and 10 μL of WST-1 reagent(Roche cat#11644807001) was added. Plates were incubated under assayconditions for 3.5 hours, centrifuged at 500×g and 75 μL supernatanttransferred to an ELISA plate (Costar cat#3369). The plates were read atOD 420-600 nm on a Spectromax 190 ELISA plate reader. The results areprovided in Table 10.

TABLE 10 HuTNFα Neutralization Assay With huTNFα Parent Antibody andDVD-Ig Constructs N-Terminal C-Terminal VD VD Parent N-TerminalC-Terminal TNFα Neu- TNFα Neu- Antibody Variable Variable tralizationtralization or DVD- Domain Domain Assay EC50 AssayEC50 Ig ID (VD) (VD)nM nM AB017 TNF 0.077 DVD224 TNF A beta (seq. 1) 0.142 DVD291 TNF RGMa0.248 DVD295 RAGE TNF 0.841 DVD296 TNF RAGE 0.158 EGFR (seq. 1) DVD289TNF 0.122

All DVD-Igs containing VDs from AB017 in either the N-terminal orC-terminal position showed neutralization in the TNFα neutralizationassay.

Example 1.1.2.H IL-6 induced pSTAT3 Assay

TF-1 cells are cultured in DMEM with 2 mM 1-glutamine, 10 mM HEPES, 100U/mL Pen/strep, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 1 mM sodiumpyruvate, 10% FBS, and 2 ng/mL GM-CSF. TF-1 cells are plated at1.5-2×10⁵ cells per well in a 10 μL volume and incubated overnight at37° C., 5% CO₂ in assay medium. (complete DMEM minus GM-CSF). Cells areplated into a 96½ well white assay plate. A 500 μg/mL working stock ofantibody (4× concentrated) is prepared in PBS. Antibodies and DVD-Igsare serial diluted 1:5 in assay medium in Marsh dilution plates. FiveuL/well of each antibody dilution is added in triplicate to the 96½ wellwhite assay plate containing the cells. Cells and antibodies or DVD-Igsare pre-incubated for 30 minutes on ice. IL-6 is prepared at 10 μg/mLstock in endotoxin free D-PBS (0.1% BSA) and a working stock of 100ng/mL (4× concentration) prepared with assay media. Five μL/well of the100 ng/mL IL-6 is added to each well. Plates are incubated for 30minutes at 37° C. Cells are lysed by adding 5 μL of 5× cell lysis bufferto all wells and the plates are shaken for 10 minutes at roomtemperature. Plates are frozen at −20° C. and the pSTAT3 SureFire Assaywas run (Perkin Elmer).

The plate is thawed at room temperature and 30 nil well of ReactionBuffer plus Activation Buffer mix containing Alpha Screen Acceptor Beeds(40 parts reaction buffer, 10 parts activation buffer and 1 partacceptor beads) is added to each well. The plate is sealed with foil toprotect it from light and is agitated gently for 2 hours at 37° C.Dilution buffer (12.5μL/well) containing Alpha Screen Donor beads (20parts dilution buffer to 1 part donor beads) is added to each well. Theplate is sealed with foil and agitated gently for 2 hours at 37° C. Theplate is brought to room temperature and read on the Alpha Screen platereader.

Example 1.1.2.1 Growth Inhibitory Effect of a Tumor Receptor MonoclonalAntibody or DVD-Igs In Vitro

Tumor receptor monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA(Dulbecco's phosphate buffered saline with 0.1% BSA) 204, are added tohuman tumor cells at final concentrations of 0.01 μg/mL-100 μg/mL in 180uL. The plates are incubated at 37° C. in a humidified, 5% CO₂atmosphere for 3 days. The number of live cells in each well isquantified using MTS reagents according to the manufacturer'sinstructions (Promega, Madison, Wis.) to determine the percent of tumorgrowth inhibition. Wells without antibody treatment are used as controlsof 0% inhibition whereas wells without cells are considered to show 100%inhibition.

Example 1.1.2.J Tumoricidal Effect of A Parent or DVD-Ig Antibody InVitro

Parent antibodies or DVD-Ig that bind to target antigens on tumor cellsmay be analyzed for tumoricidal activity. Briefly, parent antibodies orDVD-Ig are diluted in D-PBS-BSA (Dulbecco's phosphate buffered salinewith 0.1% BSA) and added to human tumor cells at final concentrations of0.01 μg/mL to 100 μg/mL 200 μL. The plates are incubated at 37° C. in ahumidified, 5% CO₂ atmosphere for 3 days. The number of live cells ineach well is quantified using MTS reagents according to themanufacturer's instructions (Promega, Madison, Wis.) to determine thepercent of tumor growth inhibition. Wells without antibody treatment areused as controls of 0% inhibition whereas wells without cells wereconsidered to show 100% inhibition.

For assessment of apoptosis, caspase-3 activation is determined by thefollowing protocol: antibody-treated cells in 96 well plates are lysedin 120 μl of 1× lysis buffer (1.67 mM Hepes, pH 7.4, 7 mM KCl, 0.83 mMMgCl₂, 0.11 mM EDTA, 0.11 mM EGTA, 0.57% CHAPS, 1 mM DTT, 1× proteaseinhibitor cocktail tablet; EDTA-free; Roche Pharmaceuticals, Nutley,N.J.) at room temperature with shaking for 20 minutes. After cell lysis,80 μl of a caspase-3 reaction buffer (48 mM Hepes, pH 7.5, 252 mMsucrose, 0.1% CHAPS, 4 mM DTT, and 20 μM Ac-DEVD-AMC substrate; BiomolResearch Labs, Inc., Plymouth Meeting, Pa.) is added and the plates areincubated for 2 hours at 37° C. The plates are read on a 1420 VICTORMultilabel Counter (Perkin Elmer Life Sciences, Downers Grove, Ill.)using the following settings: excitation=360/40, emission=460/40. Anincrease of fluorescence units from antibody-treated cells relative tothe isotype antibody control-treated cells is indicative of apoptosis.

Example 1.1.2.K Inhibition of Cell Proliferation by Parent Antibody andDVD-Ig Constructs

U87-MG human glioma tumor cells are plated at 2,000 cells/well in 100 μlin 96-well dishes in RPMI medium supplemented with 5% fetal bovineserum, and incubated at 37° C., 5% CO₂ overnight. The following day thecells are treated with serial dilutions of antibody or DVD-Igs (0.013 nMto 133 nM dose range), and incubated at 37° C. in a humidified, 5% CO₂atmosphere for 5 days. Cell survival/proliferation is measuredindirectly by assessing ATP levels using an ATPlite kit (Perkin Elmer,Waltham, Mass.) according to the manufacturer's instructions.

Example 1.1.2.L VEGF Parent Antibody and DVD-Ig Constructs PreventVEGF₁₆₅ Interaction with VEGFR1

ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) are incubated overnightat 4° C. with 100 μl PBS containing recombinant VEGFR1 extra-cellulardomain-Fc fusion protein (5 μg/ml, R&D systems, Minneapolis, Minn.).Plates are washed three times in washing buffer (PBS containing 0.05%Tween 20), and blocked for 1 hour at 25° C. in blocking buffer (PBScontaining 1% BSA). Serial dilutions of each antibody/DVD-Ig in PBScontaining 0.1% BSA are incubated with 50 μl of 2 nM biotinylated VEGFfor 1 hour at 25° C. The antibody/DVD-Ig-biotinylated VEGF mixtures (100μl) are then added to the VEGFR1-Fc coated wells and incubated at 25° C.for 10 minutes. The wells are washed three times, and then incubated for1 hour at 25° C. with 100 μl of streptavidin-HRP (KPL #474-3000,Gaithersburg, Md.). The wells are washed three times, and 100 μl ofULTRA-TMB ELISA (Pierce, Rockford, Ill.) are added per well. Followingcolor development the reaction is stopped with 1N HCL and absorbance at450 nM is measured.

Example 1.1.2.M Inhibition of Receptor Phosphorylation by ParentAntibodies or DVD-Ig Constructs In Vitro

Human carcinoma cells are plated in 96-well plates at 40,000 cells/wellin 180 μl serum-free medium (DMEM+0.1% BSA), and incubated overnight at37° C., 5% CO₂. Costar EIA plates (Lowell, Mass.) are coated with 100μl/well of receptor capture Ab (4 μg/ml final concentration), andincubated overnight at room temperature while shaking. The followingday, receptor antibody-coated ELISA plates are washed (three times withPBST=0.05% Tween 20 in PBS, pH 7.2-7.4), and 200 μl blocking solution isadded (1% BSA, 0.05% NaN3 in PBS, pH 7.2-7.4.) to block for 2 hours atroom temperature on a rocker. Human tumor cells are co-incubated withantibodies or DVD-Igs and ligand. Monoclonal antibodies or DVD-Igsdiluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1%BSA) are added to human carcinoma cells at final concentrations of 0.01μg/mL-100 μg/mL. Growth factors are simultaneously added to the cells atconcentrations of 1-100 ng/mL (200 μL), and cells are incubated at 37°C. in a humidified, 5% CO₂ atmosphere for 1 hour. Cells are lysed in 120μl/well of cold cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl,1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1% TritonX-100, 10% Glycerol, 0.1% SDS, and protease inhibitor cocktail), andincubated at 4° C. for 20 minutes with shaking. Cell lysates (100 μl)are added to the ELISA plate, and incubated overnight at 4° C. withgentle shaking. The following day, ELISA plates are washed, and 100μl/well of pTyr-HRP detection Ab is added (p-IGF1R ELISA kit, R&D System# DYC1770, Minneapolis, Minn.), and plates are incubated for 2 hours at25° C. in the dark. Plates are developed to determine phosphorylationper the manufacturer's instructions.

Example 1.1.2.N Inhibition Of VEGFR2 (KDR) Phosphorylation By VEGFParent Antibody And DVD-Ig Constructs

NIH3T3 cells expressing human VEGFR2 (KDR) are plated at 20,000cells/well (100 μl) in 96-well plates in DMEM supplemented with 10% FBS.The following day, the cells are washed twice with DMEM andserum-starved for three hours in DMEM without FBS. Anti-VEGF parentantibody or DVD-Igs (at final concentrations of 67 nM, 6.7 nM and 0.67nM) diluted in DMEM with 0.1% BSA are pre-incubated with recombinanthuman VEGF₁₆₅ (50 ng/ml) for 1 hour at 25° C. These antibody/DVD-Ig andVEGF mixtures are then added to the cells, and the plates are incubatedat 37° C. in a humidified, 5% CO₂ atmosphere for 10 minutes. Cells arewashed twice with ice cold PBS and lysed by addition of 1000 well ofCell Lysis Buffer (Cell Signaling, Boston, Mass.) supplemented with 0.1%NP40. Duplicate samples are pooled and 170 μl is added to wells of ELISAplates previously coated with anti-VEGFR2 antibody (R&D systems, AF357,Minneapolis, Minn.) and incubated at 25° C. with gentle shaking for twohours. The wells are washed five times with washing buffer (PBScontaining 0.05% Tween 20), and incubated with 50 μl of of 1:2000dilution of biotinylated anti-phosphotyrosine antibody (4G10; Millipore,Billerica, Mass.) for 1 hour at 25° C. The wells are washed five timeswith PBS containing 0.05% Tween 20, and then incubated for 1 hour at 25°C. with streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The wellsare washed three times with streptavidin-HRP (KPL #474-3000,Gaithersburg, Md.)). The wells are washed three times with PBScontaining 0.05% Tween 20, and 100 μl of ULTRA-TMB ELISA (Pierce,Rockford, Ill.) are added per well. Following color development thereaction is stopped with 1N HCL and absorbance at 450 nM was measured.

Example 1.1.2.0 Efficacy of a DVD-12 on the Growth of Human CarcinomaSubcutaneous Flank Xenografts

A-431 human epidermoid carcinoma cells are grown in vitro to 99%viability, 85% confluence in tissue culture flasks. SCID female mice(Charles Rivers Labs, Wilmington, Mass.) at 19-25 grams are injectedsubcutaneously into the right flank with 1×10⁶ human tumor cells (1:1matrigel) on study day 0. Administration (IP, QD, 3×/week) of human IgGcontrol or DVD-Ig was—initiated after mice are size matched into groupsof mice with mean tumor volumes of approximately 200 to 320 mm³ Thetumors are measured twice a week starting on approximately day 10 posttumor cell injection.

Example 1.1.2.P Binding of Monoclonal Antibodies to the Surface of HumanTumor Cell Lines as Assessed by Flow Cytometry

Stable cell lines overexpressing a cell-surface antigen of interest orhuman tumor cell lines were harvested from tissue culture flasks andresuspended in phosphate buffered saline (PBS) containing 5% fetalbovine serum (PBS/FBS). Prior to staining, human tumor cells wereincubated on ice with (100 μl) human IgG at 5 μg/ml in PBS/FCS. 1-5×10⁵cells were incubated with antibody or DVD-Ig (2 μg/mL) in PBS/FBS for30-60 minutes on ice. Cells were washed twice and 100 μl of F(ab′)2 goatanti human IgG, Fcγ-phycoerythrin (1:200 dilution in PBS) (JacksonImmunoResearch, West Grove, Pa., Cat.#109-116-170) was added. After a 30minute incubation on ice, cells were washed twice and resuspended inPBS/FBS. Fluorescence was measured using a Becton Dickinson FACSCalibur(Becton Dickinson, San Jose, Calif.).

Table 11 shows the FACS data for the DVD-Ig constructs. The geometricmean is the n root of the multiplication product of n fluorescentsignals (a1×a2×a3 . . . an). With log-transformed data the geometricmean is used to normalize the weighting of the data distribution. Thefollowing table contains the FACS geometric mean of parent antibodiesand DVD-Ig constructs.

TABLE 11  Fluorescent Activated Cell Sorting of DVD-Ig ConstructsParent  N-terminal C-terminal FACS FACS Antibody Variable VariableGeometric Geometric or DVD- Domain Domain Mean N- Mean C- Ig ID (VD)(VD) terminal terminal AB033 EGFR (SEQ2) 192.34 DVD289 TNF EGFR (SEQ2)48.84 DVD290 EGFR (SEQ2) TNF 239.34 AB001 CD20 203.47 AB033 EGFR (SEQ2)192.34 DVD236 EGFR (SEQ2) CD20 256.34

All DVDs showed binding to their cell surface targets. The N-terminaldomains of DVDs bound their targets on the cell surface as well as orbetter than the parent antibody. Binding can be restored or improved byadjusting linker length.

Example 1.1.2.Q Binding of Parent Receptor Antibody and DVD-IgConstructs to the Surface of Human Tumor Cell Lines as Assessed by FlowCytometry

Stable cell lines overexpressing cell-surface receptors or human tumorcell lines are harvested from tissue culture flasks and resuspended inDulbecco's phosphate buffered saline (DPBS) containing 1% fetal calfserum (DPBS/FCS). 1-5×10⁵ cells are incubated with 1004, antibodies orDVD-Igs (10 ug/mL) in DPBS/FCS for 30-60 minutes on ice. Cells arewashed twice and 50 μl of goat anti-human IgG-phycoerythrin (1:50dilution in DPBS/BSA) (Southern Biotech Associates, Birmingham, Ala.cat#2040-09) is added. After 30-45 minutes incubation on ice, cells arewashed twice and resuspended in 125 uL/well 1% formaldehyde in DPBS/FCS.Fluorescence was measured using a Becton Dickinson LSRII (BectonDickinson, San Jose, Calif.).

Example 1.2 Generation of Parent Monoclonal Antibodies to a HumanAntigen of Interest

Parent mouse mAbs able to bind to and neutralize a human antigen ofinterest and a variant thereof are obtained as follows:

Example 1.2.A Immunization Of Mice With a Human Antigen of Interest

Twenty micrograms of recombinant purified human antigen (e.g., IGF 1,2)mixed with complete Freund's adjuvant or Immunoeasy adjuvant (Qiagen,Valencia, Calif.) is injected subcutaneously into five 6-8 week-oldBalb/C, five C57B/6 mice, and five AJ mice on Day 1. On days 24, 38, and49, twenty micrograms of recombinant purified human antigen variantmixed with incomplete Freund's adjuvant or Immunoeasy adjuvant isinjected subcutaneously into the same mice. On day 84 or day 112 or day144, mice are injected intravenously with 1 μg recombinant purifiedhuman antigen of interest.

Example 1.2.B Generation of a Hybridoma

Splenocytes obtained from the immunized mice described in Example 1.2.Aare fused with SP2/O-Ag-14 cells at a ratio of 5:1 according to theestablished method described in Kohler, G. and Milstein (1975) Nature,256:495 to generate hybridomas. Fusion products are plated in selectionmedia containing azaserine and hypoxanthine in 96-well plates at adensity of 2.5×10⁶ spleen cells per well. Seven to ten days post fusion,macroscopic hybridoma colonies are observed. Supernatant from each wellcontaining hybridoma colonies is tested by ELISA for the presence ofantibody to the antigen of interest (as described in Example 1.1.1.A).Supernatants displaying antigen-specific activity are then tested foractivity (as described in the assays of Example 1.1.2), for example, theability to neutralize the antigen of interest in a bioassay such as thatdescribed in Example 1.1.2.1).

Example 1.2.C Identification And Characterization Of Parent MonoclonalAntibodies to a Human Target Antigen of Interest Example 1.2.C.1Analyzing Parent Monoclonal Antibody Neutralizing Activity

Hybridoma supernatants are assayed for the presence of parent antibodiesthat bind an antigen of interest, generated according to Examples 1.2.Aand 1.2.B, and are also capable of binding a variant of the antigen ofinterest (“antigen variant”). Supernatants with antibodies positive inboth assays are then tested for their antigen neutralization potency,for example, in the cytokine bioassay of Example 1.1.2.1. The hybridomasproducing antibodies with IC₅₀ values in the bioassay less than 1000 pM,in an embodiment, less than 100 pM are scaled up and cloned by limitingdilution. Hybridoma cells are expanded into media containing 10% low IgGfetal bovine serum (Hyclone #SH30151, Logan, Utah). On average, 250 mLof each hybridoma supernatant (derived from a clonal population) isharvested, concentrated and purified by protein A affinitychromatography, as described in Harlow, E. and Lane, D. 1988“Antibodies: A Laboratory Manual”. The ability of purified mAbs toinhibit the activity of its target antigen is determined, for example,using the cytokine bioassay as described in Example 1.1.2.1.

Example 1.2.C.2 Analyzing Parent Monoclonal Antibody Cross-Reactivity ToCynomolgus Target Antigen Of Interest

To determine whether the selected mAbs described herein recognizecynomolgus antigen of interest, BIACORE analysis is conducted asdescribed herein (Example 1.1.1.G) using recombinant cynomolgus targetantigen. In addition, neutralization potencies of mAbs againstrecombinant cynomolgus antigen of interest may also be measured in thecytokine bioassay (Example 1.1.2.1). MAbs with good cynocross-reactivity (in an embodiment, within 5-fold of reactivity forhuman antigen) are selected for future characterization.

Example 1.2.D Determination of the Amino Acid Sequence of the VariableRegion for Each Murine Anti-Human Monoclonal Antibody

Isolation of the cDNAs, expression and characterization of therecombinant anti-human mouse mAbs is conducted as follows. For eachamino acid sequence determination, approximately 1×10⁶ hybridoma cellsare isolated by centrifugation and processed to isolate total RNA withTrizol (Gibco BRL/Invitrogen, Carlsbad, Calif.) following manufacturer'sinstructions. Total RNA is subjected to first strand DNA synthesis usingthe SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad,Calif.) per the manufacturer's instructions. Oligo(dT) is used to primefirst-strand synthesis to select for poly(A)+RNA. The first-strand cDNAproduct is then amplified by PCR with primers designed for amplificationof murine immunoglobulin variable regions (Ig-Primer Sets, Novagen,Madison, Wis.). PCR products are resolved on an agarose gel, excised,purified, and then subcloned with the TOPO Cloning kit into pCR2.1-TOPOvector (Invitrogen, Carlsbad, Calif.) and transformed into TOP10chemically competent E. coli (Invitrogen, Carlsbad, Calif.). Colony PCRis performed on the transformants to identify clones containing insert.Plasmid DNA is isolated from clones containing insert using a QlAprepMiniprep kit (Qiagen, Valencia, Calif.). Inserts in the plasmids aresequenced on both strands to determine the variable heavy or variablelight chain DNA sequences using M13 forward and M13 reverse primers(Fermentas Life Sciences, Hanover Md.). Variable heavy and variablelight chain sequences of the mAbs are identified. In an embodiment, theselection criteria for a panel of lead mAbs for next step development(humanization) includes the following:

-   -   The antibody does not contain any N-linked glycosylation sites        (NXS), except from the standard one in CH2    -   The antibody does not contain any extra cysteines in addition to        the normal cysteines in every antibody    -   The antibody sequence is aligned with the closest human germline        sequences for VH and VL and any unusual amino acids should be        checked for occurrence in other natural human antibodies    -   N-terminal Glutamine (Q) is changed to Glutamic acid (E) if it        does not affect the activity of the antibody. This will reduce        heterogeneity due to cyclization of Q    -   Efficient signal sequence cleavage is confirmed by Mass        Spectrophotometry. This can be done with COS cell or 293 cell        material    -   The protein sequence is checked for the risk of deamidation of        Asn that could result in loss of activity    -   The antibody has a low level of aggregation    -   The antibody has solubility >5-10 mg/ml (in research phase); >25        mg/ml    -   The antibody has a normal size (5-6 nm) by Dynamic Light        Scattering (DLS)    -   The antibody has a low charge heterogeneity    -   The antibody lacks cytokine release (see Example 1.1.2.B)    -   The antibody has specificity for the intended cytokine (see        Example 1.1.2.C)    -   The antibody lacks unexpected tissue cross reactivity (see        Example 1.1.2.D)    -   The antibody has similarity between human and cynomolgus tissue        cross reactivity (see Example 1.1.2.D)

Example 1.2.2 Recombinant Humanized Parent Antibodies Example 1.2.2.1Construction And Expression Of Recombinant Chimeric Anti Human ParentAntibodies

The DNA encoding the heavy chain constant region of murine anti-humanparent mAbs is replaced by a cDNA fragment encoding the human IgG1constant region containing 2 hinge-region amino acid mutations byhomologous recombination in bacteria. These mutations are a leucine toalanine change at position 234 (EU numbering) and a leucine to alaninechange at position 235 (Lund et al., 1991, J. Immunol., 147:2657). Thelight chain constant region of each of these antibodies is replaced by ahuman kappa constant region. Full-length chimeric antibodies aretransiently expressed in COS cells by co-transfection of chimeric heavyand light chain cDNAs ligated into the pBOS expression plasmid(Mizushima and Nagata, Nucleic Acids Research 1990, Vol 18, pg 5322).Cell supernatants containing recombinant chimeric antibody are purifiedby Protein A Sepharose chromatography and bound antibody is eluted byaddition of acid buffer. Antibodies are neutralized and dialyzed intoPBS.

The heavy chain cDNA encoding a chimeric mAb is co-transfected with itschimeric light chain cDNA (both ligated in the pBOS vector) into COScells. Cell supernatant containing recombinant chimeric antibody ispurified by Protein A Sepharose chromatography and bound antibody iseluted by addition of acid buffer. Antibodies are neutralized anddialyzed into PBS.

The purified chimeric anti-human parent mAbs are then tested for theirability to bind (by Biacore) and for functional activity, e.g., toinhibit the cytokine induced production of IgE as described in Examples1.1.1.G and 1.1.2.B. Chimeric mAbs that maintain the activity of theparent hybridoma mAbs are selected for future development.

Example 1.2.2.2 Construction And Expression Of Humanized Anti HumanParent Antibodies Example 1.2.2.2.A Selection Of Human AntibodyFrameworks

Each murine variable heavy and variable light chain gene sequence isseparately aligned against 44 human immunoglobulin germline variableheavy chain or 46 germline variable light chain sequences (derived fromNCBI Ig Blast website athttp://www.ncbi.nlm.nih.gov/igblast/retrieveig.html.) using Vector NTIsoftware.

Humanization is based on amino acid sequence homology, CDR clusteranalysis, frequency of use among expressed human antibodies, andavailable information on the crystal structures of human antibodies.Taking into account possible effects on antibody binding, VH-VL pairing,and other factors, murine residues are mutated to human residues wheremurine and human framework residues are different, with a fewexceptions. Additional humanization strategies are designed based on ananalysis of human germline antibody sequences, or a subgroup thereof,that possessed a high degree of homology, i.e., sequence similarity, tothe actual amino acid sequence of the murine antibody variable regions.

Homology modeling is used to identify residues unique to the murineantibody sequences that are predicted to be critical to the structure ofthe antibody combining site, the CDRs. Homology modeling is acomputational method whereby approximate three dimensional coordinatesare generated for a protein. The source of initial coordinates andguidance for their further refinement is a second protein, the referenceprotein, for which the three dimensional coordinates are known and thesequence of which is related to the sequence of the first protein. Therelationship among the sequences of the two proteins is used to generatea correspondence between the reference protein and the protein for whichcoordinates are desired, the target protein. The primary sequences ofthe reference and target proteins are aligned with coordinates ofidentical portions of the two proteins transferred directly from thereference protein to the target protein. Coordinates for mismatchedportions of the two proteins, e.g., from residue mutations, insertions,or deletions, are constructed from generic structural templates andenergy refined to insure consistency with the already transferred modelcoordinates. This computational protein structure may be further refinedor employed directly in modeling studies. The quality of the modelstructure is determined by the accuracy of the contention that thereference and target proteins are related and the precision with whichthe sequence alignment is constructed.

For the murine mAbs, a combination of BLAST searching and visualinspection is used to identify suitable reference structures. Sequenceidentity of 25% between the reference and target amino acid sequences isconsidered the minimum necessary to attempt a homology modelingexercise. Sequence alignments are constructed manually and modelcoordinates are generated with the program Jackal (see Petrey, D. et al.(2003) Proteins 53 (Suppl. 6): 430-435).

The primary sequences of the murine and human framework regions of theselected antibodies share significant identity. Residue positions thatdiffer are candidates for inclusion of the murine residue in thehumanized sequence in order to retain the observed binding potency ofthe murine antibody. A list of framework residues that differ betweenthe human and murine sequences is constructed manually. Table 12 showsthe framework sequences chosen for this study.

TABLE 12 Sequence Of Human IgG Heavy Chain ConstantDomain And Light Chain Constant Domain SEQ ID Sequence Protein NO12345678901234567890123456789012345678901 Wild type hIgG1 58ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant regionNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Mutant hIgG1  59ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant regionNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Ig kappa  60TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK constant regionVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECIg Lambda 61 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW constant regionKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECS

The likelihood that a given framework residue would impact the bindingproperties of the antibody depends on its proximity to the CDR residues.Therefore, using the model structures, the residues that differ betweenthe murine and human sequences are ranked according to their distancefrom any atom in the CDRs. Those residues that fell within 4.5 Å of anyCDR atom are identified as most important and are recommended to becandidates for retention of the murine residue in the humanized antibody(i.e., back mutation).

In silico constructed humanized antibodies are constructed usingoligonucleotides. For each variable region cDNA, 6 oligonucleotides of60-80 nucleotides each are designed to overlap each other by 20nucleotides at the 5′ and/or 3′ end of each oligonucleotide. In anannealing reaction, all 6 oligonulceotides are combined, boiled, andannealed in the presence of dNTPs. DNA polymerase I, Large (Klenow)fragment (New England Biolabs #M0210, Beverley, Mass.) is added tofill-in the approximately 40 bp gaps between the overlappingoligonucleotides. PCR is performed to amplify the entire variable regiongene using two outermost primers containing overhanging sequencescomplementary to the multiple cloning site in a modified pBOS vector(Mizushima, S. and Nagata, S. (1990) Nucleic Acids Res. 18: 17). The PCRproducts derived from each cDNA assembly are separated on an agarose geland the band corresponding to the predicted variable region cDNA size isexcised and purified. The variable heavy region is inserted in-frameonto a cDNA fragment encoding the human IgG1 constant region containing2 hinge-region amino acid mutations by homologous recombination inbacteria. These mutations are a leucine to alanine change at position234 (EU numbering) and a leucine to alanine change at position 235 (Lundet al. (1991) J. Immunol. 147:2657). The variable light chain region isinserted in-frame with the human kappa constant region by homologousrecombination. Bacterial colonies are isolated and plasmid DNAextracted. cDNA inserts are sequenced in their entirety. Correcthumanized heavy and light chains corresponding to each antibody areco-transfected into COS cells to transiently produce full-lengthhumanized anti-human antibodies. Cell supernatants containingrecombinant chimeric antibody are purified by Protein A Sepharosechromatography and bound antibody is eluted by addition of acid buffer.Antibodies are neutralized and dialyzed into PBS.

Example 1.2.2.3 Characterization Of Humanized Antibodies

The ability of purified humanized antibodies to inhibit a functionalactivity is determined, e.g., using the cytokine bioassay as describedin Examples 1.1.2.A. The binding affinities of the humanized antibodiesto recombinant human antigen are determined using surface plasmonresonance (Biacore®) measurement as described in Example 1.1.1.B. TheIC₅₀ values from the bioassays and the affinity of the humanizedantibodies are ranked. The humanized mAbs that fully maintain theactivity of the parent hybridoma mAbs are selected as candidates forfuture development. The top 2-3 most favorable humanized mAbs arefurther characterized.

Example 1.2.2.3.A Pharmacokinetic Analysis Of Humanized Antibodies

Pharmacokinetic studies are carried out in Sprague-Dawley rats andcynomolgus monkeys. Male and female rats and cynomolgus monkeys aredosed intravenously or subcutaneously with a single dose of 4 mg/kg mAband samples are analyzed using antigen capture ELISA, andpharmacokinetic parameters are determined by noncompartmental analysis.Briefly, ELISA plates are coated with goat anti-biotin antibody (5mg/ml, 4° C., overnight), blocked with Superblock (Pierce), andincubated with biotinylated human antigen at 50 ng/ml in 10% SuperblockTTBS at room temperature for 2 hours. Serum samples are serially diluted(0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30minutes at room temperature. Detection is carried out with HRP-labeledgoat anti human antibody and concentrations are determined with the helpof standard curves using the four parameter logistic fit. Values for thepharmacokinetic parameters are determined by non-compartmental modelusing WinNonlin software (Pharsight Corporation, Mountain View, Calif.).Humanized mAbs with good pharmacokinetics profile (T1/2 is 8-13 days orbetter, with low clearance and excellent bioavailability 50-100%) areselected.

Example 1.2.2.3.B Physicochemical and In Vitro Stability Analysis OfHumanized Monoclonal Antibodies Size Exclusion Chromatography

Antibodies are diluted to 2.5 mg/mL with water and 20 mL is analyzed ona Shimadzu HPLC system using a TSK gel G3000 SWXL column (TosohBioscience, cat# k5539-05k). Samples are eluted from the column with 211mM sodium sulfate, 92 mM sodium phosphate, pH 7.0, at a flow rate of 0.3mL/minutes. The HPLC system operating conditions are the following:

Mobile phase: 211 mM Na₂SO₄, 92 mM Na₂HPO₄*7H₂O, pH 7.0

Gradient: Isocratic

Flow rate: 0.3 mL/minute

Detector wavelength: 280 nm

Autosampler cooler temp: 4° C.

Column oven temperature: Ambient

Run time: 50 minutes

Table 13 contains purity data of parent antibodies and DVD-Ig constructsexpressed as percent monomer (unaggregated protein of the expectedmolecular weight) as determined by the above protocol.

TABLE 13 Purity of Parent Antibodies and DVD-Ig Constructs as Determinedby Size Exclusion Chromatography Parent N-terminal C-terminal AntibodyVariable Variable or DVD- Domain Domain % Monomer Ig ID (VD) (VD)(purity) AB032 IL-1B 100 DVD225 A beta (seq. 1) IL-1B 86.5 DVD226 IL-1BA beta (seq. 1) 95.8 AB017 TNF 95.9 DVD223 A beta (seq. 1) TNF 91.6DVD224 TNF A beta (seq. 1) 81.7 AB017 TNF 95.9 AB059 RGMA 100 DVD291 TNFRGMA 91.9 DVD292 RGMA TNF 93.3 AB017 TNF 95.9 AB060 RAGE 91.9 DVD295RAGE TNF 94.1 DVD296 TNF RAGE 97.9 AB017 TNF 95.9 AB033 EGFR (seq. 1)100 DVD289 TNF EGFR (seq. 1) 85.4 DVD290 EGFR (seq. 1) TNF 95.6

DVD-Igs showed an excellent SEC profile with most DVD-Ig showing >90%monomer. This DVD-Ig profile is similar to that observed for parentantibodies.

SDS-PAGE

Antibodies are analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) under both reducing and non-reducingconditions. Adalimumab lot AFPO4C is used as a control. For reducingconditions, the samples are mixed 1:1 with 2× tris glycine SDS-PAGEsample buffer (Invitrogen, cat# LC2676, lot#1323208) with 100 mM DTT,and heated at 60° C. for 30 minutes. For non-reducing conditions, thesamples are mixed 1:1 with sample buffer and heated at 100° C. for 5minutes. The reduced samples (10 mg per lane) are loaded on a 12%pre-cast tris-glycine gel (Invitrogen, cat# EC6005box, lot#6111021), andthe non-reduced samples (10 mg per lane) are loaded on an 8%-16%pre-cast tris-glycine gel (Invitrogen, cat# EC6045box, lot#6111021).SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot#1351542) is used as amolecular weight marker. The gels are run in a XCe11 SureLock mini cellgel box (Invitrogen, cat# EI0001) and the proteins are separated byfirst applying a voltage of 75 to stack the samples in the gel, followedby a constant voltage of 125 until the dye front reached the bottom ofthe gel. The running buffer used is 1× tris glycine SDS buffer, preparedfrom a 10× tris glycine SDS buffer (ABC, MPS-79-080106)). The gels arestained overnight with colloidal blue stain (Invitrogen cat#46-7015,46-7016) and destained with Milli-Q water until the background is clear.The stained gels are then scanned using an Epson Expression scanner(model 1680, S/N DASX003641).

Sedimentation Velocity Analysis

Antibodies are loaded into the sample chamber of each of three standardtwo-sector carbon epon centerpieces. These centerpieces have a 1.2 cmoptical path length and are built with sapphire windows. PBS is used fora reference buffer and each chamber contained 140 μL. All samples areexamined simultaneously using a 4-hole (AN-60Ti) rotor in a BeckmanProteomeLab XL-I analytical ultracentrifuge (serial # PL106C01).

Run conditions are programmed and centrifuge control is performed usingProteomeLab (v5.6). The samples and rotor are allowed to thermallyequilibrate for one hour prior to analysis (20.0±0.1° C.). Confirmationof proper cell loading is performed at 3000 rpm and a single scan isrecorded for each cell. The sedimentation velocity conditions are thefollowing:

Sample Cell Volume: 420 mL

Reference Cell Volume: 420 mL

Temperature: 20° C.

Rotor Speed: 35,000 rpm

Time: 8:00 hours

UV Wavelength: 280 nm

Radial Step Size: 0.003 cm

Data Collection: One data point per step without signal averaging.

Total Number of Scans: 100

LC-MS Molecular Weight Measurement of Intact Antibodies

Molecular weight of intact antibodies are analyzed by LC-MS. Eachantibody is diluted to approximately 1 mg/mL with water. An 1100 HPLC(Agilent) system with a protein microtrap (Michrom Bioresources, Inc,cat#004/25109/03) is used to desalt and introduce 5 mg of the sampleinto an API Qstar pulsar i mass spectrometer (Applied Biosystems). Ashort gradient is used to elute the samples. The gradient is run withmobile phase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B(0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute.The mass spectrometer is operated at 4.5 kvolts spray voltage with ascan range from 2000 to 3500 mass to charge ratio.

LC-MS Molecular Weight Measurement of Antibody Light and Heavy Chains

Molecular weight measurement of antibody light chain (LC), heavy chain(HC) and deglycosylated HC are analyzed by LC-MS. Aantibody is dilutedto 1 mg/mL with water and the sample is reduced to LC and HC with afinal concentration of 10 mM DTT for 30 minutes at 37° C. Todeglycosylate the antibody, 100 mg of the antibody is incubated with 2mL of PNGase F, 5 mL of 10% N-octylglucoside in a total volume of 100 mLovernight at 37° C. After deglycosylation the sample is reduced with afinal concentration of 10 mM DTT for 30 minutes at 37° C. An Agilent1100 HPLC system with a C4 column (Vydac, cat#214TP5115, S/N060206537204069) is used to desalt and introduce the sample (5 mg) intoan API Qstar pulsar i mass spectrometer (Applied Biosystems). A shortgradient is used to elute the sample. The gradient is run with mobilephase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FAand 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute. The massspectrometer is operated at 4.5 kvolts spray voltage with a scan rangefrom 800 to 3500 mass to charge ratio.

Peptide Mapping

Antibody is denatured for 15 minutes at room temperature with a finalconcentration of 6 M guanidine hydrochloride in 75 mM ammoniumbicarbonate. The denatured samples are reduced with a finalconcentration of 10 mM DTT at 37° C. for 60 minutes, followed byalkylation with 50 mM iodoacetic acid (IAA) in the dark at 37° C. for 30minutes. Following alkylation, the sample is dialyzed overnight againstfour liters of 10 mM ammonium bicarbonate at 4° C. The dialyzed sampleis diluted to 1 mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mgof antibody is either digested with trypsin (Promega, cat# V5111) orLys-C(Roche, cat#11 047 825 001) at a 1:20 (w/w) trypsin/Lys-C:antibodyratio at 37° C. for 4 hrs. Digests are quenched with 1 mL of 1 N HCl.For peptide mapping with mass spectrometer detection, 40 mL of thedigests are separated by reverse phase high performance liquidchromatography (RPHPLC) on a C18 column (Vydac, cat#218TP51, S/N NE960610.3.5) with an Agilent 1100 HPLC system. The peptide separation is runwith a gradient using mobile phase A (0.02% TFA and 0.08% FA in HPLCgrade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile)at a flow rate of 50 mL/minutes. The API QSTAR Pulsar i mass spectromeris operated in positive mode at 4.5 kvolts spray voltage and a scanrange from 800 to 2500 mass to charge ratio.

Disulfide Bond Mapping

To denature the antibody, 100 mL of the antibody is mixed with 300 mL of8 M guanidine HCl in 100 mM ammonium bicarbonate. The pH is checked toensure that it is between 7 and 8 and the samples are denatured for 15minutes at room temperature in a final concentration of 6 M guanidineHCl. A portion of the denatured sample (100 mL) is diluted to 600 mLwith Milli-Q water to give a final guanidine-HCl concentration of 1 M.The sample (220 mg) is digested with either trypsin (Promega, cat #V5111, lot#22265901) or Lys-C(Roche, cat#11047825001, lot#12808000) at a1:50 trypsin or 1:50 Lys-C: antibody (w/w) ratios (4.4 mg enzyme: 220 mgsample) at 37° C. for approximately 16 hours. An additional 5 mg oftrypsin or Lys-C is added to the samples and digestion is allowed toproceed for an additional 2 hours at 37° C. Digestions are stopped byadding 1 mL of TFA to each sample. Digested samples are separated byRPHPLC using a C18 column (Vydac, cat#218TP51 S/N NE020630-4-1A) on anAgilent HPLC system. The separation is run with the same gradient usedfor peptide mapping using mobile phase A (0.02% TFA and 0.08% FA in HPLCgrade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile)at a flow rate of 50 mL/minute. The HPLC operating conditions are thesame as those used for peptide mapping. The API QSTAR Pulsar i massspectromer is operated in positive mode at 4.5 kvolts spray voltage anda scan range from 800 to 2500 mass-to-charge ratio. Disulfide bonds areassigned by matching the observed MWs of peptides with the predicted MWsof tryptic or Lys-C peptides linked by disulfide bonds.

Free Sulfhydryl Determination

The method used to quantify free cysteines in an antibody is based onthe reaction of Ellman's reagent, 5,5¢-dithio-bis (2-nitrobenzoic acid)(DTNB), with sulfhydryl groups (SH) which gives rise to a characteristicchromophoric product, 5-thio-(2-nitrobenzoic acid) (TNB). The reactionis illustrated in the formula:

DTNB+RSH®RS−TNB+TNB−+H+

The absorbance of the TNB− is measured at 412 nm using a Cary 50spectrophotometer. An absorbance curve is plotted using dilutions of 2mercaptoethanol (b-ME) as the free SH standard and the concentrations ofthe free sulfhydryl groups in the protein are determined from absorbanceat 412 nm of the sample.

The b-ME standard stock is prepared by a serial dilution of 14.2 M b-MEwith HPLC grade water to a final concentration of 0.142 mM. Thenstandards in triplicate for each concentration are prepared. Antibody isconcentrated to 10 mg/mL using an amicon ultra 10,000 MWCO centrifugalfilter (Millipore, cat# UFC801096, lot# L3KN5251) and the buffer ischanged to the formulation buffer used for adalimumab (5.57 mM sodiumphosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl,1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, pH 5.2,0.1% (w/v) Tween). The samples are mixed on a shaker at room temperaturefor 20 minutes. Then 180 mL of 100 mM Tris buffer, pH 8.1 is added toeach sample and standard followed by the addition of 300 mL of 2 mM DTNBin 10 mM phosphate buffer, pH 8.1. After thorough mixing, the samplesand standards are measured for absorption at 412 nm on a Cary 50spectrophotometer. The standard curve is obtained by plotting the amountof free SH and OD₄₁₂ nm of the b-ME standards. Free SH content ofsamples are calculated based on this curve after subtraction of theblank.

Weak Cation Exchange Chromatography

Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate, pH 6.0.Charge heterogeneity is analyzed using a Shimadzu HPLC system with aWCX-10 ProPac analytical column (Dionex, cat#054993, S/N 02722). Thesamples are loaded on the column in 80% mobile phase A (10 mM sodiumphosphate, pH 6.0) and 20% mobile phase B (10 mM sodium phosphate, 500mM NaCl, pH 6.0) and eluted at a flow rate of 1.0 mL/minute.

Oligosaccharide Profiling

Oligosaccharides released after PNGase F treatment of antibody arederivatized with 2-aminobenzamide (2-AB) labeling reagent. Thefluorescent-labeled oligosaccharides are separated by normal phase highperformance liquid chromatography (NPHPLC) and the different forms ofoligosaccharides are characterized based on retention time comparisonwith known standards.

The antibody is first digested with PNGaseF to cleave N-linkedoligosaccharides from the Fc portion of the heavy chain. The antibody(200 mg) is placed in a 500 mL Eppendorf tube along with 2 mL PNGase Fand 3 mL of 10% N-octylglucoside. Phosphate buffered saline is added tobring the final volume to 60 mL. The sample is incubated overnight at37° C. in an Eppendorf thermomixer set at 700 RPM. Adalimumab lot AFPO4Cis also digested with PNGase F as a control.

After PNGase F treatment, the samples are incubated at 95° C. for 5minutes in an Eppendorf thermomixer set at 750 RPM to precipitate outthe proteins, then the samples are placed in an Eppendorf centrifuge for2 minutes at 10,000 RPM to spin down the precipitated proteins. Thesupernatent containing the oligosaccharides are transferred to a 500 mLEppendorf tube and dried in a speed-vac at 65° C.

The oligosaccharides are labeled with 2AB using a 2AB labeling kitpurchased from Prozyme (cat# GKK-404, lot#132026). The labeling reagentis prepared according to the manufacturer's instructions. Acetic acid(150 mL, provided in kit) is added to the DMSO vial (provided in kit)and mixed by pipeting the solution up and down several times. The aceticacid/DMSO mixture (100 mL) is transferred to a vial of 2-AB dye (justprior to use) and mixed until the dye is fully dissolved. The dyesolution is then added to a vial of reductant (provided in kit) andmixed well (labeling reagent). The labeling reagent (5 mL) is added toeach dried oligosaccharide sample vial, and mixed thoroughly. Thereaction vials are placed in an Eppendorf thermomixer set at 65° C. and700-800 RPM for 2 hours of reaction.

After the labeling reaction, the excess fluorescent dye is removed usingGlycoClean S Cartridges from Prozyme (cat# GKI-4726). Prior to addingthe samples, the cartridges are washed with 1 mL of milli-Q waterfollowed with 5 ishes of 1 mL 30% acetic acid solution. Just prior toadding the samples, 1 mL of acetonitrile (Burdick and Jackson, cat#AH015-4) is added to the cartridges.

After all of the acetonitrile passed through the cartridge, the sampleis spotted onto the center of the freshly washed disc and allowed toadsorb onto the disc for 10 minutes. The disc is washed with 1 mL ofacetonitrile followed by five ishes of 1 mL of 96% acetonitrile. Thecartridges are placed over a 1.5 mL Eppendorf tube and the 2-AB labeledoligosaccharides are eluted with 3 ishes (400 mL each ish) of milli Qwater.

The oligosaccharides are separated using a Glycosep N HPLC (cat#GKI-4728) column connected to a Shimadzu HPLC system. The Shimadzu HPLCsystem consisted of a system controller, degasser, binary pumps,autosampler with a sample cooler, and a fluorescent detector.

Stability at Elevated Temperatures

The buffer of antibody is either 5.57 mM sodium phosphate monobasic,8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodiumcitrate, 6.45 mM citric acid, 66.68 mM mannitol, 0.1% (w/v) Tween, pH5.2; or 10 mM histidine, 10 mM methionine, 4% mannitol, pH 5.9 usingAmicon ultra centrifugal filters. The final concentration of theantibodies is adjusted to 2 mg/mL with the appropriate buffers. Theantibody solutions are then filter sterized and 0.25 mL aliquots areprepared under sterile conditions. The aliquots are left at either −80°C., 5° C., 25° C., or 40° C. for 1, 2 or 3 weeks. At the end of theincubation period, the samples are analyzed by size exclusionchromatography and SDS-PAGE.

The stability samples are analyzed by SDS-PAGE under both reducing andnon-reducing conditions. The procedure used is the same as describedherein. The gels are stained overnight with colloidal blue stain(Invitrogen cat#46-7015, 46-7016) and destained with Milli-Q water untilthe background is clear. The stained gels are then scanned using anEpson Expression scanner (model 1680, S/N DASX003641). To obtain moresensitivity, the same gels are silver stained using silver staining kit(Owl Scientific) and the recommended procedures given by themanufacturer is used.

Example 1.2.2.3.C Efficacy of a Humanized Monoclonal Antibody by Itselfor in Combination with Chemotherapy on the Growth of Human CarcinomaXenografts

Human cancer cells are grown in vitro to 99% viability, 85% confluencein tissue culture flasks. SCID female or male mice (Charles Rivers Labs)at 19-25 grams, are ear tagged and shaved. Mice are then inoculatedsubcutaneously into the right flank with 0.2 ml of 2×10⁶ human tumorcells (1:1 matrigel) on study day 0. Administration (IP, Q3D/week) ofvehicle (PBS), humanized antibody, and/or chemotherapy is initiatedafter mice are size matched into separate cages of mice with mean tumorvolumes of approximately 150 to 200 mm³ The tumors are measured by apair of calipers twice a week starting on approximately day 10 postinoculation and the tumor volumes calculated according to the formulaV=L×W²/2 (V: volume, mm³; L: length, mm; W: width, mm) Reduction intumor volume is seen in animals treated with mAb alone or in combinationwith chemotherapy relative to tumors in animals that received onlyvehicle or an isotype control mAb.

Example 1.2.2.3.D FACS Based Redirected Cytotoxicity (rCTL) Assay

Human CD3+ T cells were isolated from previously frozen isolatedperipheral blood mononuclear cells (PBMC) by a negative selectionenrichment column (R&D Systems, Minneapolis, Minn.; Cat.#HTCC-525). Tcells were stimulated for 4 days in flasks (vent cap, Corning, Acton,Mass.) coated with 10 μg/mL anti-CD3 (OKT-3, eBioscience, Inc., SanDiego, Calif.) and 2 μg/mL anti-CD28 (CD28.2, eBioscience, Inc., SanDiego, Calif.) in D-PBS (Invitrogen, Carlsbad, Calif.) and cultured in30 U/mL IL-2 (Roche) in complete RPMI 1640 media (Invitrogen, Carlsbad,Calif.) with L-glutamine, 55 mM β-ME, Pen/Strep, 10% FBS). T cells werethen rested overnight in 30 U/mL IL-2 before using in assay. DoHH2 orRaji target cells were labeled with PKH26 (Sigma-Aldrich, St. Louis,Mo.) according to manufacturer's instructions. RPMI 1640 media (nophenol, Invitrogen, Carlsbad, Calif.) containing L-glutamine and 10% FBS(Hyclone, Logan, Utah) was used throughout the rCTL assay. (See Dreieret al. (2002) Int J Cancer 100:690).

Effector T cells (E) and targets (T) were plated at a final cellconcentration of 10⁵ and 10⁴ cells/well in 96-well plates (Costar #3799,Acton, Mass.), respectively to give an E:T ratio of 10:1. DVD-Igmolecules were diluted to obtain concentration-dependent titrationcurves. After an overnight incubation cells are pelleted and washed withD-PBS once before resuspending in FACS buffer containing 0.1% BSA(Invitrogen, Carlsbad, Calif.), 0.1% sodium azide and 0.5 μg/mLpropidium iodide (BD) in D-PBS. FACS data was collected on a FACS CantoII machine (Becton Dickinson, San Jose, Calif.) and analyzed in Flowjo(Treestar). The percent live targets in the DVD-Ig treated samplesdivided by the percent total targets (control, no treatment) wascalculated to determine percent specific lysis. IC50s were calculated inPrism (Graphpad).

A CD3/CD20 DVD-Ig was tested for redirected toxicity and showed in vitrotumor killing with an IC50=325 pM. The sequence of this CD3/CD20 DVD-Igwas disclosed in US Patent Application Serial No. 20070071675.

Example 1.4 Generation of a DVD-Ig

DVD-Ig molecules capable of binding two antigens are constructed usingtwo parent monoclonal antibodies, one against human antigen A, and theother against human antigen B, selected as described herein.

Example 1.4.1 Generation of a DVD-Ig Having Two Linker Lengths

A constant region containing μ1 Fc with mutations at 234, and 235 toeliminate ADCC/CDC effector functions is used. Four different anti-A/BDVD-Ig constructs are generated: 2 with short linker and 2 with longlinker, each in two different domain orientations: V_(A)-V_(B)-C andV_(B)-V_(A)-C (see Table 14). The linker sequences, derived from theN-terminal sequence of human Cl/Ck or CH1 domain, are as follows:

For DVDAB constructs:

light chain (if anti-A has λ):Short linker: QPKAAP (SEQ ID NO: 15); Longlinker: QPKAAPSVTLFPP (SEQ ID NO: 16)

light chain (if anti-A has k):Short linker: TVAAP (SEQ ID NO: 13); Longlinker: TVAAPSVFIFPP (SEQ ID NO: 14)

heavy chain (γ1): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:ASTKGPSVFPLAP (SEQ ID NO: 22)

For DVDBA constructs:

light chain (if anti-B has λ):Short linker: QPKAAP (SEQ ID NO: 15); Longlinker: QPKAAPSVTLFPP (SEQ ID NO: 16)

light chain (if anti-B has k):Short linker: TVAAP (SEQ ID NO: 13); Longlinker: TVAAPSVFIFPP (SEQ ID NO: 14)

heavy chain (γ1): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:ASTKGPSVFPLAP (SEQ ID NO: 22)

Heavy and light chain constructs are subcloned into the pBOS expressionvector, and expressed in COS cells, followed by purification by ProteinA chromatography. The purified materials are subjected to SDS-PAGE andSEC analysis.

Table 14 describes the heavy chain and light chain constructs used toexpress each anti-A/B DVD-Ig protein.

TABLE 14 Anti-A/B DVD-Ig Constructs DVD-Ig Heavy chain Light chainprotein construct construct DVDABSL DVDABHC-SL DVDABLC-SL DVDABLLDVDABHC-LL DVDABLC-LL DVDBASL DVDBAHC-SL DVDBALC-SL DVDBALL DVDBAHC-LLDVDBALC-LL

Example 1.4.2 Molecular cloning of DNA constructs for DVDABSL andDVDABLL

To generate heavy chain constructs DVDABHC-LL and DVDABHC-SL, VH domainof A antibody is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VH domain of B antibody is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction. The overlapping PCR products are subcloned into Srf I andSal I double digested pBOS-hCγ1,z non-a mammalian expression vector(Abbott) by using standard homologous recombination approach.

To generate light chain constructs DVDABLC-LL and DVDABLC-SL, VL domainof A antibody is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VL domain of B antibody is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Not I double digested pBOS-hCk mammalianexpression vector (Abbott) by using standard homologous recombinationapproach. Similar approach has been used to generate DVDBASL and DVDBALLas described below:

Example 1.4.3 Molecular cloning of DNA constructs for DVDBASL andDVDBALL

To generate heavy chain constructs DVDBAHC-LL and DVDBAHC-SL, VH domainof antibody B is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VH domain of antibody A is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Sal I double digested pBOS-hCγ1,z non-amammalian expression vector (Abbott) by using standard homologousrecombination approach.

To generate light chain constructs DVDBALC-LL and DVDBALC-SL, VL domainof antibody B is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VL domain of antibody A is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Not I double digested pBOS-hCk mammalianexpression vector (Abbott) by using standard homologous recombinationapproach.

Example 1.4.4 Construction and Expression of Additional DVD-Ig Example1.4.4.1 Preparation of DVD-Ig Vector Constructs

Parent antibody amino acid sequences for specific antibodies, whichrecognize specific antigens or epitopes thereof, for incorporation intoa DVD-Ig can be obtained by preparation of hybridomas as described aboveor can be obtained by sequencing known antibody proteins or nucleicacids. In addition, known sequences can be obtained from the literature.The sequences can be used to synthesize nucleic acids using standard DNAsynthesis or amplification technologies and assembling the desiredantibody fragments into expression vectors, using standard recombinantDNA technology, for expression in cells.

For example, nucleic acid codons were determined from amino acidssequences and oligonucleotide DNA was synthesized by Blue HeronBiotechnology, Inc. (www.blueheronbio.com) Bothell, Wash. USA. Theoligonucleotides were assembled into 300-2,000 base pair double-strandedDNA fragments, cloned into a plasmid vector and sequence-verified.Cloned fragments were assembled using an enzymatic process to yield thecomplete gene and subcloned into an expression vector. (See U.S. Pat.Nos. 7,306,914; 7,297,541; 7,279,159; 7,150,969; 20080115243;20080102475; 20080081379; 20080075690; 20080063780; 20080050506;20080038777; 20080022422; 20070289033; 20070287170; 20070254338;20070243194; 20070225227; 20070207171; 20070150976; 20070135620;20070128190; 20070104722; 20070092484; 20070037196; 20070028321;20060172404; 20060162026; 20060153791; 20030215458; 20030157643).

A group of pHybE vectors (U.S. Patent Application Ser. No. 61/021,282)were used for parental antibody and DVD-Ig cloning. V1, derived frompJP183; pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVDheavy chains with a wildtype constant region. V2, derived from pJP191;pHybE-hCk V2, was used for cloning of antibody and DVD light chains witha kappa constant region. V3, derived from pJP192; pHybE-hCl V2, was usedfor cloning of antibody and DVDs light chains with a lambda constantregion. V4, built with a lambda signal peptide and a kappa constantregion, was used for cloning of DVD light chains with a lambda-kappahybrid V domain. V5, built with a kappa signal peptide and a lambdaconstant region, was used for cloning of DVD light chains with akappa-lambda hybrid V domain. V7, derived from pJP183;pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVD heavychains with a (234,235 AA) mutant constant region.

Referring to Table 15, a number of vectors were used in the cloning ofthe parent antibodies and DVD-Ig VH and VL chains.

TABLE 15 Vectors Used to Clone Parent Antibodies and DVD-Igs ID Heavychain vector Light chain vector AB001 V1 V2 AB003 V1 V2 AB010 V1 V3AB017 V1 V2 AB020 V1 V2 AB032 V1 V2 AB033 V1 V2 AB040 V1 V2 AB043 V1 V2AB044 V1 V3 AB045 V1 V2 AB046 V1 V2 AB052 V1 V2 AB059 V1 V2 AB060 V1 V2DVD193 V1 V2 DVD194 V1 V2 DVD195 V1 V2 DVD196 V1 V2 DVD197 V1 V2 DVD198V1 V2 DVD201 V1 V2 DVD202 V1 V2 DVD203 V1 V5 DVD204 V1 V4 DVD205 V1 V2DVD206 V1 V2 DVD207 V1 V2 DVD208 V1 V2 DVD209 V1 V2 DVD210 V1 V2 DVD211V1 V2 DVD212 V1 V2 DVD215 V1 V2 DVD216 V1 V2 DVD217 V1 V5 DVD218 V1 V4DVD219 V1 V2 DVD220 V1 V2 DVD221 V1 V2 DVD222 V1 V2 DVD223 V1 V2 DVD224V1 V2 DVD225 V1 V2 DVD226 V1 V2 DVD229 V1 V5 DVD230 V1 V4 DVD231 V1 V2DVD232 V1 V2 DVD233 V1 V2 DVD234 V1 V2 DVD235 V1 V2 DVD236 V1 V2 DVD239V1 V2 DVD240 V1 V2 DVD243 V1 V2 DVD244 V1 V2 DVD247 V1 V2 DVD248 V1 V2DVD255 V1 V2 DVD256 V1 V2 DVD289 V1 V2 DVD290 V1 V2 DVD291 V1 V2 DVD292V1 V2 DVD293 V1 V2 DVD294 V1 V2 DVD295 V1 V2 DVD296 V1 V2

Example 1.4.4.2 Transfection And Expression In 293 Cells

The DVD-Ig vector constructs are transfected into 293 cells forproduction of DVD-Ig protein. The 293 transient transfection procedureused is a modification of the methods published in Durocher et al.(2002) Nucleic Acids Res. 30(2):E9 and Pham et al. (2005) Biotech.Bioengineering 90(3):332-44. Reagents that were used in the transfectionincluded:

-   -   HEK 293-6E cells (human embryonic kidney cell line stably        expressing EBNA1; obtained from National Research Council        Canada) cultured in disposable Erlenmeyer flasks in a humidified        incubator set at 130 rpm, 37° C. and 5% CO₂.    -   Culture medium: FreeStyle 293 Expression Medium (Invitrogen        12338-018) plus 25 μg/mL Geneticin (G418) (Invitrogen 10131-027)        and 0.1% Pluronic F-68 (Invitrogen 24040-032).    -   Transfection medium: FreeStyle 293 Expression Medium plus 10 mM        HEPES (Invitrogen 15630-080).    -   Polyethylenimine (PEI) stock: 1 mg/mL sterile stock solution, pH        7.0, prepared with linear 25 kDa PEI (Polysciences) and stored        at less than −15° C.    -   Tryptone Feed Medium: 5% w/v sterile stock of Tryptone N1        (Organotechnie, 19554) in FreeStyle 293 Expression Medium.

Cell Preparation for Transfection:

Approximately 2-4 hours prior to transfection, HEK 293-6E cells areharvested by centrifugation and resuspended in culture medium at a celldensity of approximately 1 million viable cells per mL. For eachtransfection, 40 mL of the cell suspension is transferred into adisposable 250-mL Erlenmeyer flask and incubated for 2-4 hours.

Transfection:

The transfection medium and PEI stock are prewarmed to room temperature(RT). For each transfection, 25 μg of plasmid DNA and 50 μg ofpolyethylenimine (PEI) are combined in 5 mL of transfection medium andincubated for 15-20 minutes at RT to allow the DNA:PEI complexes toform. For the BR3-Ig transfections, 25 μg of BR3-Ig plasmid is used pertransfection. Each 5-mL DNA:PEI complex mixture is added to a 40-mLculture prepared previously and returned to the humidified incubator setat 130 rpm, 37° C. and 5% CO₂. After 20-28 hours, 5 mL of Tryptone FeedMedium is added to each transfection and the cultures are continued forsix days.

Table 16 contains the yield data for parent antibodies or DVD-Igconstructs expressed as milligrams per liter in 293 cells.

TABLE 16 Transient Expression in Yields of Parent Antibodies and DVD-IgConstructs in 293 Cells Parent N-terminal C-terminal Antibody VariableVariable Expression or DVD- Domain Domain Yield Ig ID (VD) (VD) (mg/L)AB044 A beta (seq 2) 48.8 AB017 TNF 123.2 AB044 A beta (seq 2) 48.8AB044 A beta (seq 2) 48.8 AB032 IL-1B 27 AB044 A beta (seq 2) 48.8DVD201 A beta (seq 2) A beta (seq 1) 7 DVD202 A beta (seq 1) A beta (seq2) 41.4 AB010 IGF1, 2 38.6 AB044 A beta (seq 2) 48.8 AB044 A beta (seq2) 48.8 AB040 IL-6 25.8 AB044 A beta (seq 2) 48.8 AB017 TNF 123.2 AB032IL-1B 27.0 AB010 IGF1, 2 38.6 AB040 IL-6 25.8 AB017 TNF 123.2 DVD223 Abeta (seq 1) TNF 18.2 DVD224 TNF A beta (seq 1) 30.6 AB032 IL-1B 27DVD225 A beta (seq 1) IL-1B 4.4 DVD226 IL-1B A beta (seq 1) 2.2 AB010IGF1, 2 38.6 DVD229 A beta (seq 1) IGF1, 2 64 DVD230 IGF1, 2 A beta(seq 1) 11.4 DVD231 A beta (seq 1) IL-18 11.2 DVD232 IL-18 A beta(seq 1) 18.4 AB040 IL-6 25.8 DVD233 A beta (seq 1) IL-6 5.8 AB060 RAGE107.2 DVD293 A beta (seq 1) RAGE 48.4 DVD294 RAGE A beta (seq 1) 87AB020 NGF 28 AB020 NGF 28 AB032 IL-1B 27 AB020 NGF 28 AB040 IL-6 25.8AB017 TNF 123.2 AB033 EGFR (SEQ2) 44.4 DVD289 TNF EGFR (SEQ2) 14 DVD290EGFR (SEQ2) TNF 4.8 AB017 TNF 123.2 AB060 RAGE 107.2 DVD295 RAGE TNF46.4 DVD296 TNF RAGE 39.6 AB001 CD20 90.2 AB017 TNF 123.2 AB059 RGMa17.8 DVD291 TNF RGMa 5.8 DVD292 RGMa TNF 0.6 AB001 CD20 90.2 AB033 EGFR(SEQ2) 44.4 DVD235 CD20 EGFR (SEQ2) 0 DVD236 EGFR (SEQ2) CD20 7.2

All DVDs expressed well in 293 cells. DVDs could be easily purified overa protein A column. In most cases >5 mg/L purified DVD-Ig could beobtained easily from supernatants of 293 cells.

Example 1.4.5 Characterization and Lead Selection of A/B DVD-Igs

The binding affinities of anti-A/B DVD-Igs are analyzed on Biacoreagainst both protein A and protein B. The tetravalent property of theDVD-Ig is examined by multiple binding studies on Biacore. Meanwhile,the neutralization potency of the DVD-Igs for protein A and protein Bare assessed by bioassays, respectively, as described herein. The DVD-Igmolecules that best retain the affinity and potency of the originalparent mAbs are selected for in-depth physicochemical and bio-analytical(rat PK) characterizations as described herein for each mAb. Based onthe collection of analyses, the final lead DVD-Ig is advanced into CHOstable cell line development, and the CHO-derived material is employedin stability, pharmacokinetic and efficacy studies in cynomolgus monkey,and preformulation activities.

Example 2 Generation and Characterization of Dual Variable DomainImmunoglobulins (DVD-Ig)

Dual variable domain immunoglobulins (DVD-Ig) using parent antibodieswith known amino acid sequences were generated by synthesizingpolynucleotide fragments encoding DVD-Ig variable heavy and DVD-Igvariable light chain sequences and cloning the fragments into a pHybC-D2vector according to Example 1.4.4.1. The DVD-Ig constructs were clonedinto and expressed in 293 cells as described in Example 1,4.4.2. TheDVD-Ig protein was purified according to standard methods. Functionalcharacteristics were determined according to the methods described inExample 1.1.1 and 1.1.2 as indicated. DVD-Ig VH and VL chains for theDVD-Igs of the invention are provided below.

Example 2.1 Generation of Abeta (sea. 1) and Abeta (sea. 3) DVD-Igs

TABLE 17 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 62VD193H AB044VH AB045VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPEVQLVESGGGLV QPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKNTLY LQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS 63VD193L AB044VL AB045VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPDIVMTQSPLSLPVTPGEP ASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPWTFGGGTKVEIKR 64 VD194HAB045VH AB044VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPEVKLVESGGGLVKPGGSL RLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNTLYLQMNSLR AEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 65VD194L AB045VL AB044VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPDVLVTQSPLSLPVTPGEP ASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCFQGSHVPYTFGQGTKLEIKR

Example 2.2 Generation of TNF and Abeta (sea. 2) DVD-Igs

TABLE 18 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 66VD195H AB044VH AB017VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPEVQLVESGGGLV QPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS 67 VD195L AB044VL AB017VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPDIQMTQSPSSLSASVGDR VTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQRYNRAPYTFGQGTKVEIKR 68 VD196HAB017VH AB044VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPEVKLVESGG GLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNT LYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 69 VD196L AB017VL AB044VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDVLVTQSPLSLPVTPGEPASISC RSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCFQGSHVPYTFGQGTKLEIKR

Example 2.3 Generation of IL-1beta and Abeta (sea. 2) DVD-Igs

TABLE 19 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 70VD197H AB044VH AB032VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPQVQLVESGGGVV QPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKNTLY LQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS 71 VD197L AB044VL AB032VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPEIVLTQSPDFQSVTPKEK VTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAY YCHQSSSLPFTFGPGTKVDIKR 72 VD198HAB032VH AB044VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRF TISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSASTKGPEVKLVESGGGLV KPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNTLYL QMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 73 VD198L AB032VL AB044VL EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSG TDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPDVLVTQSPLSLPVTPGEPASISC RSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCFQGSHVPYTFGQGTKLEIKR

Example 2.4 Generation of Abeta (sea. 1) and Abeta (sea. 2) DVD-Igs

TABLE 20 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 74VD201H AB044VH AB043VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPEVQLLESGGGLV QPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 75 VD201L AB044VL AB043VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPDVVMTQSPLSLPVTPGEP ASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHFPRTFGQGTKVEIKR 76 VD202HAB043VH AB044VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPEVKLVESGGGL VKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNTLY LQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 77 VD202L AB043VL AB044VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPDVLVTQSPLSLPVTPGEP ASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCFQGSHVPYTFGQGTKLEIKR

Example 2.5 Generation of IGF1,2 and Abeta (sea. 2) DVD-Igs

TABLE 21 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 78VD203H AB044VH AB010VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPQVQLVQSGAEVK KPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAY MELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS 79 VD203L AB044VL AB010VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPQSVLTQPPSVSAAPGQKV TISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEAD YYCETWDTSLSAGRVFGGGTKLTVLG 80 VD204HAB010VH AB044VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRV TMTRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSSASTKGPEVKLVESGGG LVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNTL YLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 81 VD204L AB010VL AB044VL QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKS GTSATLGITGLQTGDEADYYCETWDTSLSAGRVFGGGTKLTVLGQPKAAPDVLVTQSPLSLPVTPGEP ASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCFQGSHVPYTFGQGTKLEIKR

Example 2.6 Generation of Abeta (sea. 2) and IL-18 DVD-Igs

TABLE 22 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 82VD205H AB044VH AB046VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPEVQLVQSGTEVK KPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS 83 VD205L AB044VL AB046VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPEIVMTQSPATLSVSPGER ATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVY YCQQYNNWPSITFGQGTRLEIKR 84 VD206HAB046VH AB044VH EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQV TISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPEVKLVESGG GLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVRNT LYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 85 VD206L AB046VL AB044VL EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSG TEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPDVLVTQSPLSLPVTPGEPASIS CRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQGSHVPYTFGQGTKLEIKR

Example 2.7 Generation of IL-6 and Abeta (sea. 2) DVD-Igs

TABLE 23 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 86VD207H AB044VH AB040VH EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFT ISRDNVRNTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSSASTKGPQVTLKESGPGIL QPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSRLTISKDTSNNQV FLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSS 87 VD207L AB044VL AB040VL DVLVTQSPLSLPVTPGEPASISCRSTQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPQIVLIQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYY CQQWSGYPYTFGGGTKLEIKR 88 VD208HAB040VH AB044VH QVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSR LTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSSASTKGPEVKLVES GGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASIHNRGTIFYLDSVKGRFTISRDNVR NTLYLQMNSLRAEDTAVYYCTRGRSNSYAMDYWGQGTSVTVSS 89 VD208L AB040VL AB044VL QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGT SYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRTVAAPDVLVTQSPLSLPVTPGEPASISCR STQTLVHRNGDTYLEWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CFQGSHVPYTFGQGTKLEIKR

Example 2.8 Generation of TNF and Abeta (seq.3) DVD-Igs

TABLE 24 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 90VD209H AB045VH AB017VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGRSL RLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSL RAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS S91 VD209L AB045VL AB017VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPDIQMTQSPSSLSASVGDR VTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQRYNRAPYTFGQGTKVEIKR 92 VD210HAB017VH AB045VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPEVQLVESGG GLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKN TLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVS S93 VD210L AB017VL AB045VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDIVMTQSPLSLPVTPGEPASISC RSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCSQSTHVPWTFGGGTKVEIKR

Example 2.9 Generation of IL-1beta and Abeta (sea. 3) DVD-Igs

TABLE 25 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 94VD211H AB045VH AB032VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPQVQLVESGGGVVQPGRSL RLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKNTLYLQMNGL RAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS 95VD211L AB045VL AB032VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPEIVLTQSPDFQSVTPKEK VTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAY YCHQSSSLPFTFGPGTKVDIKR 96 VD212HAB032VH AB045VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRF TISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV QPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKNTLY LQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS 97VD212L AB032VL AB045VL EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSG TDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPDIVMTQSPLSLPVTPGEPASISC RSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCSQSTHVPWTFGGGTKVEIKR

Example 2.10 Generation of Abeta (sea. 1) and Abeta (sea. 3) DVD-Igs

TABLE 26 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234  98VD215H AB045VH AB043VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPEVQLLESGGGLVQPGGSL RLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS  99VD215L AB045VL AB043VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPDVVMTQSPLSLPVTPGEP ASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHFPRTFGQGTKVEIKR 100VD216H AB043VH AB045VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKNTL YLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS 101VD216L AB043VL AB045VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPDIVMTQSPLSLPVTPGEP ASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPWTFGGGTKVEIKR

Example 2.11 Generation of IGF1,2 and Abeta (sea. 3) DVD-Igs

TABLE 27 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 102VD217H AB045VH AB010VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSL RSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS103 VD217L AB045VL AB010VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPQSVLTQPPSVSAAPGQKV TISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEAD YYCETWDTSLSAGRVFGGGTKLTVLG 104 VD218HAB010VH AB045VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRV TMTRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSSASTKGPEVQLVESGGG LVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKNT LYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS105 VD218L AB010VL AB045VL QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKS GTSATLGITGLQTGDEADYYCETWDTSLSAGRVFGGGTKLTVLGQPKAAPDIVMTQSPLSLPVTPGEP ASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPWTFGGGTKVEIKR

Example 2.12 Generation of Abeta (sea. 3) and IL-18 DVD-Igs

TABLE 28 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 106VD219H AB045VH AB046VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPEVQLVQSGTEVKKPGESL KISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAFLQWSSL KASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVS S107 VD219L AB045VL AB046VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPEIVMTQSPATLSVSPGER ATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVY YCQQYNNWPSITFGQGTRLEIKR 108 VD220HAB046VH AB045VH EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQV TISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPEVQLVESGG GLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNAKN TLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVS S109 VD220L AB046VL AB045VL EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSG TEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPDIVMTQSPLSLPVTPGEPASIS CRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCSQSTHVPWTFGGGTKVEIKR

Example 2.13 Generation of IL-6 and Abeta (sea. 3) DVD-Igs

TABLE 29 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 110VD221H AB045VH AB040VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSSASTKGPQVTLKESGPGILQPSQTL SLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSRLTISKDTSNNQVFLKITN VDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSS 111 VD221L AB045VL AB040VL DIVMTQSPLSLPVTPGEPASISCRSSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPQIVLIQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYY CQQWSGYPYTFGGGTKLEIKR 112 VD222HAB040VH AB045VH QVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSR LTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSSASTKGPEVQLVES GGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGSTYYPDSVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS 113 VD222L AB040VL AB045VL QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGT SYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRTVAAPDIVMTQSPLSLPVTPGEPASISCR SSQSLVYSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPWTFGGGTKVEIKR

Example 2.14 Generation of TNF and Abeta (sea. 1) DVD-Igs

TABLE 30 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 114VD223H AB043VH AB017VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPEVQLVESGGGL VQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS 115 VD223L AB043VL AB017VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPDIQMTQSPSSLSASVGDR VTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQRYNRAPYTFGQGTKVEIKR 116 VD224HAB017VH AB043VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPEVQLLESGG GLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 117 VD224L AB017VL AB043VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDVVMTQSPLSLPVTPGEPASISC KSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCWQGTHFPRTFGQGTKVEIKR

Example 2.15 Generation of IL-1beta and Abeta (sea. 1) DVD-Igs

TABLE 31 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 118VD225H AB043VH AB032VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPQVQLVESGGGV VQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKNTL YLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS 119 VD225L AB043VL AB032VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPEIVLTQSPDFQSVTPKEK VTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAY YCHQSSSLPFTFGPGTKVDIKR 120 VD226HAB032VH AB043VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRF TISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSASTKGPEVQLLESGGGLV QPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 121 VD226L AB032VL AB043VL EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSG TDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPDVVMTQSPLSLPVTPGEPASISC KSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCWQGTHFPRTFGQGTKVEIKR

Example 2.16 Generation of IGF1,2 and Abeta (sea. 1) DVD-Igs

TABLE 32 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 122VD229H AB043VH AB010VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPQVQLVQSGAEV KKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTA YMELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS 123 VD229L AB043VL AB010VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPQSVLTQPPSVSAAPGQKV TISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEAD YYCETWDTSLSAGRVFGGGTKLTVLG 124 VD230HAB010VH AB043VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRV TMTRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSSASTKGPEVQLLESGGG LVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 125 VD230L AB010VL AB043VL QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKS GTSATLGITGLQTGDEADYYCETWDTSLSAGRVFGGGTKLTVLGQPKAAPDVVMTQSPLSLPVTPGEP ASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHFPRTFGQGTKVEIKR

Example 2.17 Generation of Abeta (sea. 1) and IL-18 DVD-Igs

TABLE 33 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 126VD231H AB043VH AB046VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPEVQLVQSGTEV KKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTA FLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS 127 VD231L AB043VL AB046VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPEIVMTQSPATLSVSPGER ATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVY YCQQYNNWPSITFGQGTRLEIKR 128 VD232HAB046VH AB043VH EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQV TISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPEVQLLESGG GLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 129 VD232L AB046VL AB043VL EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSG TEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPDVVMTQSPLSLPVTPGEPASIS CKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPRTFGQGTKVEIKR

Example 2.18 Generation of IL-6 and Abeta (sea. 1) DVD-Igs

TABLE 34 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 130VD233H AB043VH AB040VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPQVTLKESGPGI LQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSRLTISKDTSNNQ VFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSS 131 VD233L AB043VL AB040VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPQIVLIQSPAIMSASPGEK VTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYY CQQWSGYPYTFGGGTKLEIKR 132 VD234HAB040VH AB043VH QVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSR LTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSSASTKGPEVQLLES GGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 133 VD234L AB040VL AB043VL QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGT SYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRTVAAPDVVMTQSPLSLPVTPGEPASISCK SSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CWQGTHFPRTFGQGTKVEIKR

Example 2.19 Generation of Abeta (sea. 1) and RAGE DVD-Igs

TABLE 35 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 134VD293H AB060VH AB043VH QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGYINTNTGESIYSEEFKGRF AFSLETSASTAYLQINNLKNEDTATYFCARSRMVTAYGMDYWGQGTSVTVSSASTKGPEVQLLESGGG LVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS 135 VD293L AB060VL AB043VL DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQRPGQSPKLLIFSASNRYTGVPDRFTGSGSG TDFTLTLSNMQPEDLADYFCQQYSSYPLTFGVGTKLELKRTVAAPDVVMTQSPLSLPVTPGEPASISC KSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCWQGTHFPRTFGQGTKVEIKR 136 VD294HAB043VH AB060VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPQIQLVQSGPEL KKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGYINTNTGESIYSEEFKGRFAFSLETSASTA YLQINNLKNEDTATYFCARSRMVTAYGMDYWGQGTSVTVSS 137 VD294L AB043VL AB060VL DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPDIVMTQSQKFMSTSVGDR VSITCKASQNVGTAVAWYQQRPGQSPKLLIFSASNRYTGVPDRFTGSGSGTDFTLTLSNMQPEDLADY FCQQYSSYPLTFGVGTKLELKR

Example 2.20 Generation of NGF and IL-18 DVD-Igs

TABLE 36 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 138VD239H AB046VH AB020VH EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQV TISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSSASTKGPQVQLQESGP GLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQ FSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS 139 VD239L AB046VL AB020VL EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSG TEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT CRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ EHTLPYTFGQGTKLEIKR 140 VD240H AB020VHAB046VH QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVT ISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPEVQLVQSGT EVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFN TAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS 141 VD240L AB020VL AB046VL DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPEIVMTQSPATLSVSPGERATLSC RASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQY NNWPSITFGQGTRLEIKR

Example 2.21 Generation of NGF and IL-1beta DVD-Igs

TABLE 37 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 142VD243H AB032VH AB020VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRF TISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSASTKGPQVQLQESGPGLV KPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSL KLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS 143 VD243L AB032VL AB020VL EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSG TDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPDIQMTQSPSSLSASVGDRVTITC RASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQE HTLPYTFGQGTKLEIKR 144 VD244H AB020VHAB032VH QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVT ISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPQVQLVESGG GVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKN TLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS 145 VD244L AB020VL AB032VL DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPEIVLTQSPDFQSVTPKEKVTITC RASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQS SSLPFTFGPGTKVDIKR

Example 2.22 Generation of NGF and IL-6 DVD-Igs

TABLE 38 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 146VD247H AB040VH AB020VH QVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPSGKGLEWLAHIYWDEDKRYNPSLKSR LTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFDYWGQGTTLTVSSASTKGPQVQLQES GPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSK NQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS 147 VD247L AB040VL AB020VLQIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYW YQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTK LEIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKR 148 VD248H AB020VH AB040VHQVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDL NWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWY ATSYYFDYWGQGTLVTVSSASTKGPQVTLKESGPGILQPSQTLSLTCSFSGFSLSTNGMGVSWIRQPS GKGLEWLAHIYWDEDKRYNPSLKSRLTISKDTSNNQVFLKITNVDTADTATYYCARRRIIYDVEDYFD YWGQGTTLTVSS 149 VD248L AB020VLAB040VL DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPQIVLIQSPAIMSASPGEKVTMTC SASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWS GYPYTFGGGTKLEIKR

Example 2.23 Generation of TNF and EGFR (see. 2) DVD-Igs

TABLE 39 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 150VD289H AB017VH AB033VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ VFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA 151 VD289L AB017VL AB033VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDILLTQSPVILSVSPGERVSFSC RASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN NNWPTTFGAGTKLELKR 152 VD290H AB033VHAB017VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLS INKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPEVQLVESGGGL VQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS 153 VD290L AB033VL AB017VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSG TDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPDIQMTQSPSSLSASVGDRVTITC RASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRY NRAPYTFGQGTKVEIKR

Example 2.24 Generation of TNF and RAGE DVD-Igs

TABLE 40 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 154VD295H AB060VH AB017VH QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGYINTNTGESIYSEEFKGRF AFSLETSASTAYLQINNLKNEDTATYFCARSRMVTAYGMDYWGQGTSVTVSSASTKGPEVQLVESGGG LVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS 155 VD295L AB060VL AB017VL DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQRPGQSPKLLIFSASNRYTGVPDRFTGSGSG TDFTLTLSNMQPEDLADYFCQQYSSYPLTFGVGTKLELKRTVAAPDIQMTQSPSSLSASVGDRVTITC RASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRY NRAPYTFGQGTKVEIKR 156 VD296H AB017VHAB060VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPQIQLVQSGP ELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGYINTNTGESIYSEEFKGRFAFSLETSAS TAYLQINNLKNEDTATYFCARSRMVTAYGMDYWGQGTSVTVSS 157 VD296L AB017VL AB060VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDIVMTQSQKFMSTSVGDRVSITC KASQNVGTAVAWYQQRPGQSPKLLIFSASNRYTGVPDRFTGSGSGTDFTLTLSNMQPEDLADYFCQQY SSYPLTFGVGTKLELKR

Example 2.25 Generation of CD-20 and EGFR (see. 1) DVD-Igs

TABLE 41 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 158VD255H AB001VH AB003VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPQVQLQESGP GLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSK TQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS 159 VD255L AB001VL AB003VL QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTITCQ ASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFD HLPLAFGGGTKVEIKR 160 VD256H AB003VHAB001VH QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSR LTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPQVQLQQPGAEL VKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTA YMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA 161 VD256L AB003VL AB001VL DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSG TDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPQIVLSQSPAILSPSPGEKVTMTC RASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWT SNPPTFGGGTKLEIKR

Example 2.26 Generation of CD-20 and EGFR (see. 2) DVD-Igs

TABLE 42 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 162VD235H AB001VH AB033VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKA TLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ VFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA 163 VD235L AB001VL AB033VL QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPDILLTQSPVILSVSPGERVSFSCR ASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNN NWPTTFGAGTKLELKR 164 VD236H AB033VHAB001VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLS INKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPQVQLQQPGAEL VKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTA YMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA 165 VD236L AB033VL AB001VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSG TDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPQIVLSQSPAILSPSPGEKVTMTC RASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWT SNPPTFGGGTKLEIKR

Example 2.27 Generation of RGMA and TNF DVD-Igs

TABLE 43 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 1234567890123456789012345678901234 166VD291H AB017VH AB059VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPEVQLVESGG GLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKN TLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSS 167 VD291L AB017VL AB059VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPDVVLTQTPVSLSVTLGDQASMSC RSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVY YCFQATHDPLTFGSGTKLEIKR 168 VD292HAB059VH AB017VH EVQLVESGGGLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRF TISRDNSKNTLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSSASTKGPEVQLVESGGGLVQPG RSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS 169 VD292L AB059VL AB017VL DVVLTQTPVSLSVTLGDQASMSCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFI GSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIKRTVAAPDIQMTQSPSSLSASVGDR VTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQRYNRAPYTFGQGTKVEIKR

Example 2.28 Cloning Vector Sequences Used to Clone Parent Antibody andDVD-Ig Sequences

TABLE 44 SEQ ID Vector Nucleotide sequences NO name123456789012345678901234567890123456789012345678901 170 V1GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGCGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC 171 V2ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCACCATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC 172 V3CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGACTTGGACCCCACTCCTCTTCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG 173 V4ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCACCATGACTTGGACCCCACTCCTCTTCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG 174 V5CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC 175 V7GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGCGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC

The present invention incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

-   Ausubel et al. (eds.), Current Protocols in Molecular Biology, John    Wiley &Sons, NY (1993);-   Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).-   Controlled Drug Bioavailability, Drug Product Design and    Performance, Smolen and Ball (eds.), Wiley, New York (1984);-   Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids    and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford    University Press, New York, N.Y., (1999);-   Goodson, in Medical Applications of Controlled Release, vol. 2, pp.    115-138 (1984);-   Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas    563-681 (Elsevier, N.Y., 1981;-   Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor    Laboratory Press, 2nd ed. 1988);-   Kabat et al., Sequences of Proteins of Immunological Interest    (National Institutes of Health, Bethesda, Md. (1987) and (1991);-   Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological    Interest, Fifth Edition, U.S. Department of Health and Human    Services, NIH Publication No. 91-3242;-   Kontermann and Dubel eds., Antibody Engineering (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Kriegler, Gene Transfer and Expression, A Laboratory Manual,    Stockton Press, NY (1990);-   Lu and Weiner eds., Cloning and Expression Vectors for Gene Function    Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp.    (ISBN 1-881299-21-X).-   Medical Applications of Controlled Release, Langer and Wise (eds.),    CRC Pres., Boca Raton, Fla. (1974);-   Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An    Introduction To Genetic Engineering (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V. 2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6).-   Sustained and Controlled Release Drug Delivery Systems, J. R.    Robinson, ed., Marcel Dekker, Inc., New York, 1978-   Winnacker, E.L. From Genes To Clones: Introduction To Gene    Technology (1987) VCH Publishers, NY (translated by Horst    Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety, as are the references cited therein. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

1-76. (canceled)
 77. A binding protein comprising first and secondpolypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first variable domain; VD2 is a second variable domain;C is a constant domain; X1 is a linker; X2 is an Fc region; n is 0 or 1,wherein the VD1 domains on the first and second polypeptide chains forma first functional target binding site and the VD2 domains on the firstand second polypeptide chains form a second functional target bindingsite, and wherein the binding protein is capable of binding TumorNecrosis Factor α (TNF-α) and Repulsive Guidance Molecule Family MemberA (RGMA), wherein 1) the variable domains that form a functional targetbinding site for TNF-α comprise CDRs 1-3 from SEQ ID NO: 34 and CDRs 1-3from SEQ ID NO: 35; and 2) the variable domains that form a functionaltarget binding site for RGMA comprise CDRs 1-3 from SEQ ID NO: 54 andCDRs 1-3 from SEQ ID NO:
 55. 78. A binding protein comprising first andsecond polypeptide chains, each independently comprisingVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain; VD2 is asecond variable domain; C is a constant domain; X1 is a linker; X2 is anFc region; n is 0 or 1, wherein the VD1 domains on the first and secondpolypeptide chains form a first functional target binding site and theVD2 domains on the first and second polypeptide chains form a secondfunctional target binding site, and wherein the binding protein iscapable of binding Tumor Necrosis Factor α (TNF-α) and RepulsiveGuidance Molecule Family Member A (RGMA), wherein 1) the variabledomains that form a functional target binding site for TNF-α compriseSEQ ID NO: 34 and/or SEQ ID NO: 35; and 2) the variable domains thatform a functional target binding site for RGMA comprise SEQ ID NO: 54and/or SEQ ID NO:
 55. 79. The binding protein of claim 77, comprisingfirst and second polypeptide chains, wherein a. the first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a firstheavy chain variable domain; VD2 is a second heavy chain variabledomain; C is a heavy chain constant domain; X1 is a first linker; X2 isan Fc region; n is 0 or 1; and b. the second polypeptide chain comprisesa second VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chainvariable domain; VD2 is a second light chain variable domain; C is alight chain constant domain; X1 is a second linker; n is 0 or 1 for(X1)n; and n is 0 for (X2)n.
 80. The binding protein of claim 77,wherein the first and second polypeptide chains comprise: SEQ ID NO: 166and SEQ ID NO: 167, or SEQ ID NO: 168 and SEQ ID NO:
 169. 81. Thebinding protein of claim 80, comprising: DVD291 (comprising SEQ ID NO:166 and SEQ ID NO: 167), or DVD292 (comprising SEQ ID NO: 168 and SEQ IDNO: 169).
 82. The binding protein of claim 77, comprising two firstpolypeptide chains and two second polypeptide chains and four functionalbinding sites.
 83. The binding protein of claim 77, wherein the bindingprotein is capable of binding to: a. TNF-α with an EC₅₀ of at most775.32 nM as measured by direct binding ELISA, with a K_(D) of at most2.21×10⁻⁹ M as measured by surface plasmon resonance, or with an EC₅₀ ofat most 0.248 nM as measured by HuTNFα neutralization assay, and/or b.RGMA with an EC₅₀ of at most 2.16 nM as measured by direct binding ELISAor with a K_(D) of at most 1.30×10⁻⁸ M as measured by surface plasmonresonance.
 84. The binding protein of claim 77, wherein the bindingprotein is a crystallized binding protein.
 85. The binding protein ofclaim 77, wherein the Fc region is a variant sequence Fc region.
 86. Thebinding protein of claim 77, wherein the Fc region is an IgG1, IgG2,IgG3, IgG4, IgA, IgM, IgE, or IgD.
 87. A binding protein conjugatecomprising the binding protein of claim 77, the binding proteinconjugate further comprising an immunoadhesion molecule, an imagingagent, a therapeutic agent, or a cytotoxic agent.
 88. The bindingprotein conjugate of claim 87, wherein the imaging agent is aradiolabel, an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, or biotin.
 89. The bindingprotein conjugate of claim 88, wherein the radiolabel is ³H, ¹⁴C, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.
 90. The bindingprotein conjugate of claim 87, wherein the therapeutic agent or thecytotoxic agent is an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, a toxin, or an apoptotic agent.91. A pharmaceutical composition comprising the binding protein of claim77 and a pharmaceutically acceptable carrier.
 92. The pharmaceuticalcomposition of claim 91, further comprising at least one additionaltherapeutic agent selected from the group consisting of an imagingagent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor,a co-stimulation molecule blocker, an adhesion molecule blocker, ananti-cytokine antibody or functional fragment thereof, methotrexate,cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNFantagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, and a cytokine antagonist.
 93. A method oftreating a subject for a disease or a disorder, the method comprisingthe step of administering to the subject the binding protein of claim 77such that treatment is achieved.
 94. The method of claim 93, wherein thedisease or disorder is selected from the group consisting of rheumatoidarthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis,Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, Addison's disease, sporadic, polyglandular deficiency type Iand polyglandular deficiency type II, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia areata, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, atheromatous disease/arteriosclerosis, atopic allergy,autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus,pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombspositive haemolytic anaemia, acquired pernicious anaemia, juvenilepernicious anaemia, myalgic encephalitis/Royal Free Disease, chronicmucocutaneous candidiasis, giant cell arteritis, primary sclerosinghepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiencyrelated diseases, hepatitis B, hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease, Sjögren'sdisease associated lung disease, ankylosing spondylitis associated lungdisease, vasculitic diffuse lung disease, haemosiderosis associated lungdisease, drug-induced interstitial lung disease, fibrosis, radiationfibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia,lymphocytic infiltrative lung disease, postinfectious interstitial lungdisease, gouty arthritis, autoimmune hepatitis, type-1 autoimmunehepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmunehepatitis (anti-LKM antibody hepatitis), autoimmune mediatedhypoglycaemia, type B insulin resistance with acanthosis nigricans,hypoparathyroidism, acute immune disease associated with organtransplantation, chronic immune disease associated with organtransplantation, osteoarthrosis, primary sclerosing cholangitis,psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmuneneutropaenia, renal disease NOS, glomerulonephritides, microscopicvasulitis of the kidneys, lyme disease, discoid lupus erythematosus,male infertility idiopathic or NOS, sperm autoimmunity, multiplesclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertensionsecondary to connective tissue disease, Goodpasture's syndrome,pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever,rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjörgren'ssyndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia,idiopathic thrombocytopaenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis, vitiligo acute liver disease,chronic liver diseases, alcoholic cirrhosis, alcohol-induced liverinjury, choleosatatis, idiosyncratic liver disease, drug-inducedhepatitis, non-alcoholic steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders, depression,schizophrenia, Th2 Type and Th1 Type mediated diseases, acute andchronic pain, different forms of pain, cancers, lung cancer, breastcancer, stomach cancer, bladder cancer, colon cancer, pancreatic cancer,ovarian cancer, prostate cancer, rectal cancer, hematopoieticmalignancies, leukemia, lymphoma, Abetalipoprotemia, acrocyanosis, acuteand chronic parasitic or infectious processes, acute leukemia, acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute orchronic bacterial infection, acute pancreatitis, acute renal failure,adenocarcinomas, aerial ectopic beats, AIDS dementia complex,alcohol-induced hepatitis, allergic conjunctivitis, allergic contactdermatitis, allergic rhinitis, allograft rejection, alpha-l-antitrypsindeficiency, amyotrophic lateral sclerosis, anemia, angina pectoris,anterior horn cell degeneration, anti-CD3 therapy, antiphospholipidsyndrome, anti-receptor hypersensitivity reactions, aortic andperipheral aneuryisms, aortic dissection, arterial hypertension,arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation(sustained or paroxysmal), atrial flutter, atrioventricular block, Bcell lymphoma, bone graft rejection, bone marrow transplant (BMT)rejection, bundle branch block, Burkitt's lymphoma, burns, cardiacarrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,cardiopulmonary bypass inflammation response, cartilage transplantrejection, cerebellar cortical degenerations, cerebellar disorders,chaotic or multifocal atrial tachycardia, chemotherapy associateddisorders, chronic myelocytic leukemia (CML), chronic alcoholism,chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL),chronic obstructive pulmonary disease (COPD), chronic salicylateintoxication, colorectal carcinoma, congestive heart failure,conjunctivitis, contact dermatitis, cor pulmonale, coronary arterydisease, Creutzfeldt-Jakob disease, culture negative sepsis, cysticfibrosis, cytokine therapy associated disorders, dementia pugilistica,demyelinating diseases, dengue hemorrhagic fever, dermatitis,dermatologic conditions, diabetes, diabetes mellitus, diabeticateriosclerotic disease, diffuse Lewy body disease, dilated congestivecardiomyopathy, disorders of the basal ganglia, Down's syndrome inmiddle age, drug-induced movement disorders induced by drugs which blockCNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis,endocarditis, endocrinopathy, epiglottitis, epstein-barr virusinfection, erythromelalgia, extrapyramidal and cerebellar disorders,familial hematophagocytic lymphohistiocytosis, fetal thymus implantrejection, Friedreich's ataxia, functional peripheral arterialdisorders, fungal sepsis, gas gangrene, gastric ulcer, glomerularnephritis, graft rejection of any organ or tissue, gram negative sepsis,gram positive sepsis, granulomas due to intracellular organisms, hairycell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hayfever, heart transplant rejection, hemachromatosis, hemodialysis,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,hemorrhage, hepatitis A, His bundle arrythmias, HIV infection/HIVneuropathy, Hodgkin's disease, hyperkinetic movement disorders,hypersensitity reactions, hypersensitivity pneumonitis, hypertension,hypokinetic movement disorders, hypothalamic-pituitary-adrenal axisevaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis,antibody mediated cytotoxicity, Asthenia, infantile spinal muscularatrophy, inflammation of the aorta, influenza a, ionizing radiationexposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusioninjury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinalmuscular atrophy, Kaposi's sarcoma, kidney transplant rejection,legionella, leishmaniasis, leprosy, lesions of the corticospinal system,lipedema, liver transplant rejection, lymphederma, malaria, malignamtlymphoma, malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmultisystem disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), mycobacterium aviumintracellulare, mycobacterium tuberculosis, myelodyplastic syndrome,myocardial infarction, myocardial ischemic disorders, nasopharyngealcarcinoma, neonatal chronic lung disease, nephritis, nephrosis,neurodegenerative diseases, neurogenic muscular atrophies, neutropenicfever, non-hodgkins lymphoma, occlusion of the abdominal aorta and itsbranches, occulsive arterial disorders, okt3 therapy,orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, progressive supranucleopalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, seniledementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, subacute sclerosingpanencephalitis, syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, valvular heartdiseases, varicose veins, vasculitis, venous diseases, venousthrombosis, ventricular fibrillation, viral and fungal infections, vitalencephalitis/aseptic meningitis, vital-associated hemaphagocyticsyndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograftrejection of any organ or tissue, acute coronary syndromes, acuteidiopathic polyneuritis, acute inflammatory demyelinatingpolyradiculoneuropathy, acute ischemia, adult Still's disease,anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia,atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmunedisorder associated with streptococcus infection, autoimmuneenteropathy, autoimmune hearing loss, autoimmune lymphoproliferativesyndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarianfailure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovasculardisease, catastrophic antiphospholipid syndrome, celiac disease,cervical spondylosis, chronic ischemia, cicatricial pemphigoid,clinically isolated syndrome (cis) with risk for multiple sclerosis,childhood onset psychiatric disorder, dacryocystitis, dermatomyositis,diabetic retinopathy, disk herniation, disk prolaps, drug induced immunehemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythemamultiforme, erythema multiforme major, gestational pemphigoid,Guillain-Barré syndrome (GBS), Hughes syndrome, idiopathic Parkinson'sdisease, idiopathic interstitial pneumonia, IgE-mediated allergy, immunehemolytic anemia, inclusion body myositis, infectious ocularinflammatory disease, inflammatory demyelinating disease, inflammatoryheart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis,keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier disease,Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis,macular degeneration, microscopic polyangiitis, morbus bechterev, motorneuron disorders, mucous membrane pemphigoid, multiple organ failure,myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve rootdisorders, neuropathy, non-A non-B hepatitis, optic neuritis,osteolysis, pauciarticular JRA, peripheral artery occlusive disease(PAOD), peripheral vascular disease (PVD), peripheral artery, disease(PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa),polychondritis, poliosis, polyarticular JRA, polyendocrine deficiencysyndrome, polymyositis, polymyalgia rheumatica (PMR), primaryParkinsonism, prostatitis, pure red cell aplasia, primary adrenalinsufficiency, recurrent neuromyelitis optica, restenosis, rheumaticheart disease, sapho (synovitis, acne, pustulosis, hyperostosis, andosteitis), secondary amyloidosis, shock lung, scleritis, sciatica,secondary adrenal insufficiency, silicone associated connective tissuedisease, sneddon-wilkinson dermatosis, spondilitis ankylosans,Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmicretinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumornecrosis factor receptor, type 1 allergic reaction, type II diabetes,urticaria, usual interstitial pneumonia (UIP), vasculitis, vernalconjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKHsyndrome), wet macular degeneration, and wound healing.
 95. The methodof claim 93, wherein the administering is parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolical,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardial, intraperitoneally, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,by bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.
 96. An isolated nucleic acid encoding the binding proteinof claim
 77. 97. A vector comprising the isolated nucleic acid of claim96.
 98. A host cell comprising the vector of claim
 97. 99. The host cellof claim 98, wherein the host cell is selected from the group consistingof a prokaryotic cell, an Escherichia coli cell, a eukaryotic cell, ananimal cell, a plant cell, a fungal cell, a mammalian cell, a yeastcell, an avian cell, an insect cell, a CHO cell, a COS cell, anSaccharomyces cerevisiae cell, and an Sf9 cell.
 100. A method ofproducing a binding protein, the method comprising the step of culturingthe host cell of claim 97 in culture medium under conditions sufficientto produce the binding protein.
 101. A method of determining thepresence, amount or concentration of TNF-α and/or RGMA in a test sampleby an immunoassay, wherein the method comprises the step of contactingthe test sample with at least one binding protein and at least onedetectable label, and wherein the at least one binding protein comprisesthe binding protein of claim
 77. 102. The method of claim 101, furthercomprising the steps of: contacting the test sample with the at leastone binding protein, wherein the binding protein binds to an epitope onTNF-α and/or RGMA so as to form a first complex; contacting the complexwith the at least one detectable label, wherein the detectable labelbinds to the binding protein or an epitope on TNF-α and/or RGMA that isnot bound by the binding protein to form a second complex; and detectingthe presence, amount, or concentration of TNF-α and/or RGMA in the testsample based on the signal generated by the detectable label in thesecond complex, wherein the presence, amount, or concentration of TNF-αand/or RGMA is directly correlated with the signal generated by thedetectable label.
 103. The method of claim 101, further comprising thesteps of: contacting the test sample with the at least one bindingprotein, wherein the binding protein binds to an epitope on TNF-α and/orRGMA so as to form a first complex; contacting the complex with the atleast one detectable label, wherein the detectable label competes withTNF-α and/or RGMA for binding to the binding protein so as to form asecond complex; and detecting the presence, amount, or concentration ofTNF-α and/or RGMA in the test sample based on the signal generated bythe detectable label in the second complex, wherein the presence,amount, or concentration of TNF-α and/or RGMA is indirectly correlatedwith the signal generated by the detectable label.
 104. A kit forassaying a test sample for the presence, amount, or concentration ofTNF-α and/or RGMA, the kit comprising a. instructions for assaying thetest sample for TNF-α and/or RGMA, and b. at least one binding proteincomprising the binding protein of claim 77.